Role of Metabolic Activation and the TRPA1 Receptor in the Sensory Irritation Response to Styrene and Naphthalene

Abstract

The current study was aimed at examining the role of cytochrome P450 (CYP450) activation and the electrophile-sensitive transient receptor potential ankyrin 1 receptor (TRPA1) in mediating the sensory irritation response to styrene and naphthalene. Toward this end, the sensory irritation to these vapors was measured in female C57Bl/6J mice during 15-min exposure via plethysmographic measurement of the duration of braking at the onset of each expiration. The sensory irritation response to 75 ppm styrene and 7 ppm naphthalene was diminished threefold or more in animals pretreated with the CYP450 inhibitor metyrapone, providing evidence of the role of metabolic activation in the response to these vapors. The sensory irritation response to styrene (75 ppm) and naphthalene (7.6 ppm) was virtually absent in TRPA1−/− knockout mice, indicating the critical role of this receptor in mediating the response. Thus, these results support the hypothesis that styrene and naphthalene vapors initiate the sensory irritation response through TRPA1 detection of their CYP450 metabolites.

Sensory irritation represents a common response to inspired volatile organic compounds. This response is mediated by nasal trigeminal nerves and, in the human, is characterized by tickling, itching, and/or painful nasal sensations (Alarie, 1973; Nielsen et al., 2007). This response forms the basis for a large number of occupational exposure levels and also represents a common complaint of indoor air quality (Hall et al., 1993; Hodgson, 2002). In mouse models, the sensory irritation response is characterized by a reduced breathing frequency due to a braking at the onset of each expiration (Alarie, 1973; Desesa et al., 2008; Larsen et al., 2009; Morris et al., 2003; Vijayaraghavan et al., 1993). This response can be quantified either by measurement of the reduction in breathing frequency during irritant exposure or by measurement of the duration of braking at the onset of each expiration. The duration of braking can be increased from baseline values of approximately 10 ms to values well over 500 ms, depending on the irritant and exposure concentration (Morris et al., 2005).

The molecular basis for sensory nerve activation has been the subject of much research. Recent studies have revealed the potential importance of the transient receptor potential ankyrin 1 receptor (TRPA1). TRPA1 is sensitive to several oxidants and electrophiles including acrolein, allyl isothiocyanate, chlorine, 4-hydroxynonenal, and hydrogen peroxide (Bautista et al., 2006; Bessac and Jordt, 2008; Bessac et al., 2008, 2009), raising the potential that many oxidant and/or electrophilic sensory irritants stimulate trigeminal sensory nerves via this pathway. Nasal tissues of rodents express high levels of cytochromes P450 (CYP450), biotransformation enzymes that can result in the formation of electrophilic metabolites from volatile organic compound vapors (Ding and Dahl, 2003; Ding and Kaminsky, 2003; Thornton-Manning and Dahl, 1997). Two such vapors are styrene and naphthalene. Nasal dosimetry studies have revealed that both are extensively metabolized within nasal mucosa (Morris, 2000; Morris and Buckpitt, 2009). The nasal cytotoxicity of these vapors is thought to be due to the formation of epoxide metabolites within the nose by CYP2F (Buckpitt et al., 2002; Cruzan et al., 2002; Lee et al., 2005). Styrene and naphthalene are metabolized by CYP2F2 in the mouse, CYP2F4 in the rat, and CYP2F1 in the human, albeit at a much lower rate (Baldwin et al., 2005; Cruzan, 2008; Cruzan et al., 2009). Styrene is metabolized in both the olfactory and the respiratory mucosa of the mouse nasal cavity (Green et al., 2001). Information on the regional distribution of naphthalene metabolism in the nose of the mouse is not available; however, in the rat the specific activity of respiratory mucosa is 40-fold lower than that in the olfactory mucosa (Lee et al., 2005).

In addition to being nasal cytotoxicants, styrene and naphthalene are also sensory irritants, raising the possibility that the stimulation of trigeminal nerves may be reflective of TRPA1 activation by their epoxide metabolites. To investigate this hypothesis, two series of experiments were performed. First, the sensory irritation responses to styrene and naphthalene were measured via plethysmography in mice with and without pretreatment with the CYP450 inhibitor metyrapone (Morris 1993, 2000). The sensory irritation response to styrene and naphthalene was next measured in wild-type and TRPA1−/− knockout mice to assess the role of this receptor. Finally, in vitro studies on nasal metabolism of naphthalene in respiratory and olfactory nasal mucosa were also performed. Were metabolic activation a prerequisite for sensory irritation, then the distribution of metabolic activity within the nose may provide insights into the location of the trigeminal nerve endings that are stimulated by these vapors.

MATERIALS AND METHODS

Animals and reagents.

All mice were obtained from Jackson Laboratories (Bar Harbor, ME). Female C57Bl/6J wild-type mice were used in the metabolism and CYP450 inhibition studies. The TRPA1−/− mouse studies used commercially available female B6;129P-Trpa1<tm1Kykw>/J and age-matched female control mice. TRPA1 functionality in these mice was examined by challenge with the known TRPA1 agonist vapor acrolein (Bautista et al., 2006). Animals were acclimated at least 10 days prior to use and were 8–16 weeks of age at the time of use. Mice were housed over hardwood shavings (Sani-Chip Dry, P.J. Murphy Forest Products, Montville, NJ) in animal rooms maintained at 22°C–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. When administered, the CYP450 inhibitor, metyrapone, was given ip at a dose of 150 mg/kg (25 mg/ml in distilled water). All protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee. Styrene (reagent grade 98% purity), naphthalene (reagent grade 99% purity), and metyrapone were obtained from Sigma-Aldrich (St Louis, MO). All other agents were obtained from local suppliers and were the highest purity available.

In vitro studies.

Metabolism rates of naphthalene were studied in vitro in whole nose, respiratory, or olfactory mucosal homogenates. Nasal tissues were obtained by dissection in urethane-anesthetized (1.3 g/kg ip) mice and prepared in 2–4 ml of Krebs-Ringer buffer. Crude homogenates were spun at 10,000 × g for 5 min and the supernatants used in the assay. For assay, homogenates were incubated with 100μM reduced nicotinamide adenine dinucleotide phosphate (NADPH) and naphthalene for 10 min at 37°C in sealed glass vials. The total incubation volume was 0.5 ml, incubation vial contained ∼0.15 and 0.45 mg protein for the respiratory and olfactory mucosa samples, respectively. Naphthalene was added at a total volume of 5 μl from stock solutions prepared in ethanol. The reaction was stopped by the addition of an equal volume of n-heptane. The resultant heptane layer was analyzed via high-performance liquid chromatography (HPLC) using a Supelco C18 column and a mobile phase of 65:35 acetonitrile:water and monitored at a wavelength of 280 nm. Disappearance rates of parent naphthalene and appearance rates of metabolite were linear with time and protein concentration. Standard curves were prepared with naphthalene as well as for the metabolites 1-naphthol and 2-naphthol.

In vivo studies.

Sensory irritation was monitored by plethysmography. Spontaneously breathing mice were placed in a Buxco double plethysmograph (Buxco, Inc., Sharon, CT) for a 10-min baseline period followed by a 15-min exposure period. Breathing parameters were measured during these times using the Buxco noninvasive mechanics software. Animals were restrained by a latex collar but were not anesthetized. Mice were able to withdraw their heads from the headspace (exposure chamber) side of the plethysmograph at any time but did not. Breathing frequency and the duration of braking at the onset of expiration were measured. These duration of braking values were obtained by the Buxco software by analysis of the transducer-derived pressure signals from the plethysmograph. For duration of braking, the Buxco signal was reprocessed to provide duration of braking at the onset of expiration measured as the time required to achieve 20% of the peak expiratory flow for each breath (Desesa et al., 2008).

Atmosphere generation/analysis.

Mice were exposed to target concentrations of 75 ppm styrene and 7 ppm naphthalene. These were selected based on pilot studies to produce a marked sensory irritant response. Styrene and acrolein atmospheres were generated by passing the liquid into a heated flask; air passing through the flask was then diluted and fed into the plethysmograph. Naphthalene atmospheres were generated by passing air over crystalline naphthalene in three flasks in series at a temperature of 23°C. Chamber air temperature was between 22°C and 25°C, and relative humidity was approximately 50%. Vapor concentrations were measured during each exposure on air samples drawn every 3 min from the headspace of the plethysmograph. Samples were injected into a 0.5-ml gas-sampling loop of a Varian 3600 gas chromatograph (Varian, Sugarland, TX) equipped with flame ionization detector using a 15M DB-WAX megabore column (Agilent Technologies, Santa Clara, CA). Standard curves were prepared by volatilizing known amounts of styrene, naphthalene, or acrolein in 4-l glass containers and injecting air samples into the gas-sampling loops. Exposure air concentrations were calculated on the basis of peak areas and the standard curves.

Data analysis.

Data are presented as mean ± SD unless otherwise indicated. Data were log transformed prior to statistical analysis as appropriate to correct for heteroscedasticity. Comparison among groups was made by repeated measures ANOVA with the repeated measure being time. If appropriate, this was followed by Newman-Keuls tests to determine at which times significant effects were observed. All statistical calculations were performed with Statistica Software (StatSoft, Tulsa, OK). A p value of 0.05 or less was required for statistical significance.

RESULTS

In Vivo Studies

To assess the role of CYP450 activation, the effect of pretreatment with the CYP450 inhibitor, metyrapone, on the sensory irritation response to styrene and naphthalene was examined. Shown in Figures 1A and 1B are the duration of braking responses to styrene and naphthalene, respectively, in control and metyrapone-pretreated mice. The measured exposure concentrations were 77 ppm (styrene) and 6.9 ppm (naphthalene). Data for each irritant were analyzed separately by repeated measures ANOVA with the factors being pretreatment and time. In control and metyrapone-pretreated mice, the duration of braking during baseline averaged less than 25 ms. In control mice, the duration of braking was significantly elevated over baseline levels throughout the entire styrene exposure. The peak duration of braking response occurred during minute 10 of exposure and averaged 560 ± 272 ms. The average duration of braking throughout the entire styrene exposure was 330 ms. In metyrapone-pretreated mice, the duration of braking during styrene exposure was significantly increased over baseline during the entire exposure; the peak response occurred at the end of the exposure and was 140 ms, a value fourfold less than that in control mice. The average duration of braking during the entire exposure in metyrapone-pretreated mice was 95 ms. The duration of braking in metyrapone-pretreated mice was significantly lower than that in control mice during minutes 2–15 of exposure. The CYP inhibitor 5-phenyl-1-pentyne was not used extensively in this study because it reduced the sensory irritation of a nonmetabolized vapor (capsaicin) by about a third; however, it is interesting to note that this inhibitor completely abolished the sensory irritation response to styrene (data not shown).

FIG. 1.

(A) Shown is the time of braking during baseline (−9 to 0 min) and exposure (0–15 min) to 75 ppm styrene in control and metyrapone-pretreated mice. Data are presented as mean ± SD; there were five to six mice per group. Duration of braking was significantly increased over baseline values in control mice throughout the entire exposure. Asterisks indicate the times at which the response in the metyrapone-pretreated mice was significantly lower than that in control mice. The solid bar indicates the times of exposure at which the duration of braking in metyrapone-pretreated mice was significantly greater than baseline. (B) Shown is the time of braking during baseline (−9 to 0 min) and exposure (0–15 min) to 7 ppm naphthalene in control and metyrapone-pretreated mice. Data are presented as mean ± SD; there were five to six mice per group. Duration of braking was significantly increased over baseline values in control mice throughout the entire exposure. Asterisks indicate the times at which the response in the metyrapone-pretreated mice was significantly lower than that in control mice. At no time during the exposure was the duration of braking in metyrapone-pretreated mice significantly greater than baseline.

In control mice, the duration of braking was increased over baseline levels throughout the entire exposure to naphthalene (Fig. 1B). The peak duration of braking response occurred during minute 6 of exposure and averaged 760 ± 360 ms. The naphthalene exposure concentration averaged 6.9 ppm. The average duration of braking throughout the naphthalene exposure was 450 ms. At no time did the duration of braking significantly exceed baseline levels in metyrapone-pretreated mice indicating that the drug blocked the sensory irritation response to naphthalene. The duration of braking in metyrapone-pretreated mice was significantly lower than that in control mice during the entire exposure period.

To assess the role of the TRPA1, the sensory irritation response to styrene and naphthalene was measured in wild-type and TRPA1−/− (knockout) mice. Absence of TRPA1 functionality was confirmed by challenging mice with the known TRPA1 agonist acrolein (see below). Shown in Figures 2A and 2B are the duration of braking responses to styrene (average concentration 75 ppm) and naphthalene (average concentration 7.6 ppm), respectively, in these strains. Data for each irritant were analyzed separately by repeated measures ANOVA with the factors being strain and time. Preexposure duration of braking averaged less than 25 ms and was similar in wild-type and TRPA1−/− strains. In wild-type mice, the duration of braking during styrene exposure was significantly higher than that in control throughout the entire exposure; the peak response of 570 ± 250 ms occurred in minute 13. The average duration of braking throughout the entire exposure was 330 ms. In TRPA1−/− mice, the duration of braking was significantly higher than baseline only during the first minute of exposure at which time it averaged 49 ms. The duration of braking in TRPA1−/− mice was significantly lower than that in control mice during minutes 2–15 of exposure.

FIG. 2.

(A) Shown is the time of braking during baseline (−9 to 0 min) and exposure (0–15 min) to 75 ppm styrene in wild-type and TRPA1−/− mice. Data are presented as mean ± SD; there were four mice per group. Duration of braking was significantly increased over baseline values in wild-type mice throughout the entire exposure. Asterisks indicate the times at which the response in the TRPA1−/− mice was significantly lower than that in wild-type mice. The solid bar indicates the times of exposure at which the duration of braking TRPA1−/− mice was significantly greater than baseline. (B) Shown is the time of braking during baseline (−9 to 0 min) and exposure (0–15 min) to 7 ppm naphthalene in wild-type and TRPA1−/− mice. Data are presented as mean ± SD; there were four mice per group. Duration of braking was significantly increased over baseline values in wild-type mice throughout the entire exposure. Asterisks indicate the times at which the response in the TRPA1−/− mice was significantly lower than that in wild-type mice. The solid bar indicates the times of exposure at which the duration of braking in metyrapone-pretreated mice was significantly greater than baseline.

In wild-type mice, the duration of braking was increased over baseline levels throughout the entire exposure to naphthalene (Fig. 2B). The peak duration of braking response occurred during minute 6 of exposure and averaged 1000 ± 390 ms. The average duration of braking throughout the naphthalene exposure was 620 ms. In TRPA1−/− mice, the duration of braking was significantly higher than baseline only during the first minute of exposure at which time it averaged 34 ms. The duration of braking in TRPA1−/− mice was significantly lower than that in wild-type mice during minutes 2–15 of exposure.

TRPA1 functionality was assessed by examining the sensory irritant response to the known TRPA1 agonist acrolein at an exposure concentration of 1.0 ppm. This concentration was based on the previous studies (Morris et al., 2003). Because acrolein exposure might modulate nasal function, this challenge was performed 2 weeks after the studies described above. Acrolein challenge produced a moderate sensory irritation response in wild-type mice with the peak duration of braking being 317 ms (minute 6 of exposure) and the average duration of braking throughout the exposure being 260 ms. One of the purchased TRPA1−/− mice exhibited a response to acrolein challenge (peak 140 ms, average 40 ms duration of braking, respectively); data from this mouse were excluded. No response (duration of braking no higher than control) was seen in any other TRPA1−/− mouse confirming the absence of acrolein sensitivity and by inference, the absence of TRPA1 functionality.

In Vitro Studies

In initial studies, the nasal metabolism of naphthalene was measured in whole-nose homogenates at naphthalene substrate concentrations of 6.25, 25, 100, or 200μM. A more polar metabolite (earlier eluting) was observed on HPLC that had a retention time similar to that of 1- or 2-naphthol (these were not resolved under the HPLC conditions). The appearance of this peak was time dependent and NADPH dependent. It was quantitated on the basis of the average molar extinction coefficient for 1- and 2-napthol as determined by daily standard curves. Based on the results, an estimate for the K_m (nonlinear regression) was 20μM. At this concentration, the metabolism rates, as calculated by appearance of product and that calculated by disappearance of parent naphthalene, were quantitatively similar.

Specific activities for naphthalene metabolism were then determined at a substrate concentration of 100μM in both olfactory and respiratory mucosa homogenates. On a whole-tissue basis, the activity in olfactory mucosa averaged 16 ± 4 nmol/min-tissue compared to 3.4 ± 0.7 nmol/min-tissue in respiratory mucosa (data shown as mean ± SEM, n = 4). On a per milligram basis, the activities in the olfactory and respiratory mucosa were 36 ± 9 and 23 ± 5 nmol/min-mg, respectively. Thus, the average activity in olfactory mucosa was two- to fourfold higher than that in respiratory mucosa.

DISCUSSION

The overall goal of the current study was to examine the importance of metabolic activation and the TRPA1 in the sensory irritation response to two volatile organic compounds (styrene and naphthalene) that are substrates for nasal CYP450. Nasal dosimetry studies have shown that inhaled styrene is extensively metabolized in the mouse nose (Morris, 2000). Nasal metabolism of styrene has been well investigated in vitro (Green et al., 2001). It is primarily metabolized by CYP2F, and the primary metabolites are side chain and ring epoxides. CYP450 activity is present in both respiratory and olfactory mucosa of the mouse, with the levels being approximately twofold higher in the latter tissue. Local CYP450-dependent metabolic activation is thought to be a critical step in the toxic response of the nose to styrene (Cruzan et al., 2002). The nasal cytotoxicity of naphthalene is also thought to be due to the formation of a CYP450 metabolite (Buckpitt et al., 2002; Genter et al., 2006). Like styrene, nasal dosimetry studies have shown that inhaled naphthalene is extensively metabolized in the rat nose (Morris and Buckpitt, 2009) and pilot studies indicate that the same is true for naphthalene dosimetry in the mouse nose (Morris, unpublished observations).

In the rat, CYP450 metabolism of naphthalene occurs primarily by CYP2F4 (Baldwin et al., 2005) and results in epoxide formation that can then spontaneously and/or enzymatically (epoxide hydrolase) be converted to 1-napthol, 2-napthol, and/or 1,2-dihydro-1,2-dihydroxynaphthalene. The specific activity for naphthalene metabolism in the rat olfactory mucosa may be 40 times higher than that in respiratory mucosa (Buckpitt et al., 2002; Lee et al., 2005). The goals of the current metabolism studies were quite limited; they were to confirm that mouse nasal tissues extensively metabolized naphthalene and to provide an estimate of the specific activity in respiratory and olfactory mucosae. The current studies revealed that nasal metabolism of naphthalene was NADPH dependent and resulted in peaks consistent with the formation of 1- or 2-naphthol, known CYP450 metabolites of naphthalene (Buckpitt et al., 2002). The specific activity on a whole-nose basis was 20 nmol/min-whole nose. At a minute ventilation rate of 25 ml/min and a concentration of 7 ppm, a mouse inhales 7 nmol/min, a value less than the specific activity. Thus, it can be appreciated that the metabolic capacity of the mouse nose is large relative to the inspired burden. The total activity in the olfactory mucosa exceeded that in the respiratory mucosa by fourfold. The difference between olfactory and respiratory metabolic activity for naphthalene is fairly similar to that observed for styrene in the mouse nose (Green et al., 2001) and dissimilar to the 40-fold ratio observed for naphthalene in the rat nose (Lee et al., 2005).

Both styrene and naphthalene are known nasal CYP450 substrates (Buckpitt et al., 2002; Green et al., 2001), and the sensory irritation response to both vapors was greatly diminished in animals pretreated with the CYP450 inhibitor metyrapone, providing strong evidence that the sensory irritation response was metabolite dependent. In the case of naphthalene, the sensory irritation response was absent in metyrapone-pretreated animals. A minimal sensory irritation response to styrene was observed in metyrapone-pretreated mice suggesting that either the degree of inhibition of styrene was not complete or a small role exists for the parent compound, styrene, in the sensory irritation response. Further studies are needed to examine this possibility. Metabolic activity for both styrene and naphthalene is higher in olfactory than respiratory mucosa, by a factor of roughly two- to fourfold. Given the role of metabolic activation, these results suggest that trigeminal nerve endings in the olfactory mucosa may be responsible for the sensory irritation response but that respiratory mucosal endings may participate as well. CYP450 expression in the nose is not associated with sensory nerves but is expressed in adjacent cells (Dahl and Hadley, 1991; Ding and Dahl, 2003). Thus, it is likely that the active metabolites diffuse from their site of formation to interact with trigeminal nerve endings and/or cause the release of paracrine mediators from the cells in which they are formed which then stimulate trigeminal nerve endings (Vaughan et al., 2006). Our previous studies (Stanek et al., 2001) have shown that the sensory nerve–mediated vasodilatory response to acetaldehyde in the rat is diminished by pretreatment with the aldehyde dehydrogenase inhibitor cyanamide, thus implicating a role for the metabolite, acetic acid, in nasal sensory nerve activation. Thus, it is possible that metabolic activation may contribute to the sensory neuronal response to a variety of irritants.

The TRPA1 is a member of the transient receptor potential family and appears to be important in the detection and activation of sensory nerves by numerous electrophilic and/or oxidant compounds (Bessac and Jordt, 2008). This includes acrolein, allyl isothiocyanate, chlorine, hydrogen peroxide, and 4-hydroxynonanal (Andre et al., 2008; Bautista et al., 2006; Bessac and Jordt, 2008; Bessac et al., 2008). Given its sensitivity to oxidants/electrophiles, the TRPA1 is a likely candidate for initiation of sensory nerve responses by the CYP450 metabolites of styrene and naphthalene. Except for the first minute of exposure, the sensory irritation response to styrene and naphthalene was absent in TRPA1−/− mice. The TRPA1 is expressed on trigeminal C fibers (Bessac and Jordt, 2008). The virtual absence of response to styrene and naphthalene in the TRPA1 knockout mice provides strong evidence that these vapors activate the sensory irritation response through trigeminal C fibers that express this receptor.

Interestingly, TRPA1−/− mice demonstrated a small (< 40 ms) transient (1-min) duration of braking response to both vapors at the onset of exposure. This may well have occurred in wild-type mice but gone undetected due to the overall magnitude of the sensory irritation response. This small transient response is similar to that induced by adenosine aerosol via nasal trigeminal Aδ fiber activation (Vaughan et al., 2006). Perhaps, styrene and naphthalene are acting in this way. Alternatively, during the first minute of exposure mice exhibit exploratory behavior and other behaviors (Desesa et al., 2008), and this small transient response to styrene and naphthalene may be reflective of metabolite modifying such behaviors as evidenced by the fact it was not observed in metyrapone-pretreated mice. Future studies may clarify the initial small transient response. Since this transient response was observed in the TRPA1−/− mice it is not mediated via the same mechanism as the prolonged large sensory irritation response that occurs throughout the 15-min exposure.

The current results support the hypothesis that metabolic activation by CYP450 and detection of metabolites by TRPA1 are responsible for the sensory irritation responses to styrene and naphthalene. The nasal cavity is known to express multiple CYP450 enzymes. A wide array of volatile organic compounds are substrates for these enzymes (Dahl and Lewis, 1993; Ding and Dahl, 2003; Ding and Kaminsky, 2003; Thornton-Manning and Dahl, 1997), and the metabolites are electrophilic. Thus, it is possible that CYP450 metabolism to a electrophilic species followed by interaction with TRPA1 may be a common pathway for volatile organic compound activation of the sensory irritation response.

The sensory irritant potential is often assessed in the mouse (Kuwabara et al., 2007; Schapper, 1993) and is often quantified by the RD50, the concentration that produces a 50% decrease in the breathing frequency. This exposure level is thought to be intolerable in the human (Kuwabara et al., 2007), but empirical data suggest that, in the absence of other information, an occupational exposure level set at 3% of the mouse RD50 may protect against sensory irritation in the human (Kuwabara et al., 2007; Schapper, 1993). Styrene is clearly discordant in this regard; the data compiled by Kuwabara et al. (2007) indicate that the lowest observable effect level for nasal irritation by styrene in the human is equal or higher than the RD50 in the mouse. Unlike mouse tissues, CYP450 activation of styrene in human nasal tissues was undetectable (Green et al., 2001). Thus, the species difference in metabolism provides an explanation of this discordance. Empirical relationships between mouse RD50 levels and human responsiveness are based on compilations of data from numerous vapors (e.g., Kuwabara et al., 2007; Schapper, 1993); perhaps, these correlations could be refined by excluding metabolically activated compounds. Structure activity relationships for induction of sensory irritation have been proposed based on the chemical structure of the parent vapor (Abraham et al., 1998; Alarie et al., 1998). The currents studies suggest that for some volatile organic compounds, these relationships may be more complex. If metabolites are responsible for sensory nerve activation, then structure activity relationships for the parent compound might better incorporate metabolic potential. This may be particularly true if metabolic activation and TRPA1-based activation of sensory nerves by CYP450 metabolites is a common mechanism by which volatile organic compounds initiate the sensory irritation response.