Effect of Oral Cavity Loci and Cultural Background on Responses to Capsaicin

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Effect of oral cavity loci and cultural background on responses to capsaicin

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By Danica N Berry Graduate Program in Food Science and Technology

The Ohio State University 2020

Master's Examination Committee: Dr. Christopher T. Simons, Advisor Dr. M. Monica Giusti Dr. Devin Peterson

Copyrighted by Danica N Berry 2020

Abstract

Capsaicin is a chemesthetic compound found in chili peppers that activates the receptor

TRPV1 to elicit painful, burning sensations. Responses to capsaicin can be greatly decreased, or desensitized, following repeated exposure. Although the TRPV1 receptor is expressed ubiquitously throughout the human oral cavity, there is not a clear understanding of the behavior of many oral tissues in response to capsaicin.

Additionally, the effect of cultural aspects outside of chili pepper affinity on capsaicin responses has not been clearly determined. Therefore, the goals of the present study were to (1) investigate capsaicin irritation and desensitization across oral cavity mucosae and

(2) determine capsaicin sensitivity differences between cultures. Specifically, the first two aims were to determine differences in perception and characterize desensitization over time on the tongue, cheek, hard palate, and lip. The third study aim was to determine sensitivity differences across Caucasian American and Indian cultures by using a design controlling for chili pepper affinity.

Caucasian Americans and Indians were recruited for Experiment 1 and completed a survey questionnaire. A chili pepper use and liking score (CPULS) was calculated for each potential subject recruit based on the survey responses. Participants were selected based on matching of chili pepper use frequencies and average CPULS between groups.

To determine sensitivity on each oral cavity area, irritation intensity of a capsaicin stimulus (100 ppm) was measured over a 10-minute period. Area under the time-intensity curve (AUC), peak intensity, and time to peak intensity parameters were dependent

ii variables derived from these ratings. Data were analyzed using ANOVA with sex, frequency of use, and oral cavity areas as factors. To characterize desensitization, capsaicin stimuli were applied to the capsaicin- and control-pretreated locations and subjects completed 2-AFC (“Which side feels stronger?”) questions followed by intensity ratings over a 3.5-minute period. Binomial statistics and paired t-tests were used to analyze the data.

Oral mucosal location was found to impact perceptual responses. Specifically, max intensity and overall intensity were greater on the tongue compared to hard palate, and the hard palate compared to cheek and lip, but did not differ between the cheek and lip.

Time to reach max intensity was greater on the hard palate than on the tongue, cheek, and lip, but not among the latter three areas. Capsaicin desensitization investigated from the second task was clear on the tongue and hard palate but was not evident on the cheek and lip. Based on the lack of response differentiation and desensitization found on the cheek and lip in Experiment 1, a follow-up experiment was conducted. A greater capsaicin concentration (1000ppm) was utilized in Experiment 2 in order to evaluate the cheek and the lip.

Results from Experiment 2, which utilized a higher capsaicin concentration, showed that the responses on the lip were greater than on the cheek, and desensitization was observable on both areas. However, desensitization was observed to a lesser extent on the cheek than the lip.

The Caucasian American and Indian sample groups in Experiment 1 were well matched and did not differ in age, frequency of consumption, or chili pepper use and liking score

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(CPULS) (p’s>0.05). Results demonstrated that, after controlling for these external factors, there were no significant effects of cultural group, sex, or frequency of chili pepper use on AUC or peak intensity.

This research demonstrates that oral cavity areas differ in their responsiveness over time to capsaicin and the extent of perceivable desensitization. The findings here likely serve as indicators of differing sensitivity across the tissues which may be the result of epithelial tissue type and receptor density differences.

The controlled design of this study which tightly matched subject cohorts also allowed us to determine that there were no sensitivity differences to capsaicin between Indians and

Caucasian Americans. These data support that differences in responsiveness to capsaicin may be the result of controlled factors such as chili pepper use and liking rather than other cultural attributes.

Acknowledgments

I would first like to thank my advisor, Dr. Chris Simons. I truly value the advice, support, and freedom to pursue my interests that you have provided me with over the past two years. I could not have asked for a better mentor!

I would also like to thank my fellow Simons lab members and peers for encouraging me throughout this entire process. This research could not have gone forward without your insight and help from the beginning of the process. I would also like to thank my family and friends for consistently believing in me.

Finally, I would like to thank Dr. Giusti and Dr. Peterson for serving on my committee and being a part of my journey.

Vita

2014 ...... Lakeridge High School, OR 2018 ...... B.S. Food Science and Technology, Oregon State University 2018 to present ...... Graduate Research Fellow, Department of Food Science and Technology, The Ohio State University

Fields of Study

Major Field: Food Science and Technology

Table of Contents

Abstract ...... ii

Acknowledgments ...... v

Vita ...... vi

List of Tables ...... x

List of Figures ...... xi

Chapter 1: Literature review...... 1

1.1 The Oral Cavity …………………………….…………………………………………1

1.1.1 Tongue, Cheek, Lip, and Hard Palate Mucosa…………………..………….2

1.2 Capsaicin: Physical and Chemical Properties…………………………………………5

1.3 Neurobiology & Pharmacology of Capsaicin…………………………………………6

1.3.1 Desensitization of the Capsaicin Receptor…………………….……………7

1.4 Psychophysical Capsaicin Research in the Oral Cavity………………………………8

1.4.1 Sensitization, Desensitization, and Stimulus-Induced Recovery……………9

1.4.2 Modulation by Other Chemesthetic Stimuli……………………………….11

1.4.3 Capsaicin Perception Across Non-Lingual Areas………………………….12

1.5 Importance and Use of Chili Pepper Among Cultures……………………………....13

1.6 Use of Capsaicin as a Model of Chili Pepper Irritancy…………………………..….14

1.6.1 Capsaicin Sensitivity and Chili Pepper Consumption……………………..15

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Chapter 2: Assessing regional sensitivity and desensitization to capsaicin among oral cavity mucosae…………………………………………………………………………...17

2.1 Abstract………………………………………………………………………………17

2.2 Introduction…………………………………………………………………………..18

2.3 Experiment 1…………………………………………………………………………20

2.3.1 Methods…………………………………………………………………….20

2.4.1.1 Subjects…………………………………………………………..20

2.4.1.2 Stimuli……………………………………………………………21

2.4.1.3 Procedure………………………………………………………...21

2.4.1.4 Data Analysis…………………………………………………….24

2.3.2 Results……………………………………………………………………...25

2.4 Experiment 2…………………………………………………………………………35

2.4.1 Methods…………………………………………………………………….35

2.4.1.1 Subjects…………………………………………………………..35

2.4.1.2 Stimuli……………………………………………………………36

2.4.1.3 Procedure………………………………………………………...36

2.4.1.4 Data Analysis…………………………………………………….36

2.3.2 Results……………………………………………………………………...37

2.5 Discussion……………………………………………………………………………40

Chapter 3: Determination of capsaicin sensitivity differences between Caucasian American and Indian cultural groups: A study designed to control for chili pepper affinity……………………………………………………………………………………47 viii

3.1 Abstract………………………………………………………………………………47

3.2 Introduction…………………………………………………………………………..48

3.3 Methods………………………………………………………………………………50

3.3.1 Subjects…………………………………………………………………….50

3.3.2 Survey Questionnaire Related to Chili Pepper Use and Liking……………51

3.3.3 Stimuli……………………………………………………………………...52

3.3.4 Procedure…………………………………………………………………..52

3.3.5 Data Analysis………………………………………………………………55

3.4 Results………………………………………………………………………………..56

3.5 Discussion………….………………………………………………………………...61

Chapter 4: Overall conclusions…………………………………………………………64

Reference List…………………………………………………………………………...66

Appendix A: Consent Form……………………………………………………………..74

Appendix B: Subject Questionnaire………………………………………………….....80

List of Tables

Table 2.1 Sex, age, chili pepper consumption frequency of subjects in Experiment 1…26

Table 3.1 Sex, age, chili pepper consumption frequency, age began consuming chili peppers, and CPULS between Caucasian American and Indian subject groups………...57

List of Figures

Figure 1.1 Branches of the mandibular nerve. The mandibular nerve that originates from the trigeminal nerve innervate the tongue, cheek, and lip mucosa via the lingual, buccal, and inferior alveolar nerve branches, respectively..………………………...... 4

Figure 1.2 Branches of the maxillary nerve. The maxillary nerve branches into the greater palatine and nasopalatine nerves to supply the mucosa of the hard palate………..5

Figure 1.3 Capsaicin Chemical Structure. Taken from PubChem………………………..6

Figure 1.4 Capsaicin sensitization and desensitization on the human tongue. Single application of capsaicin followed by a 15-minute rest period resulted in desensitization. Repeated exposures (5, 10, or 15) resulted in sensitization followed by desensitization after the 15-minute rest period. From Green (2000)……..………………………………11

Figure 2.1 (A) Mean max log irritation intensity. (B) Mean area under time intensity curve (AUC). After initial vehicle and capsaicin (100ppm) stimuli application, subjects rated the intensity of irritation on each side over a 10-minute period. Means of individual max irritation intensity ratings and area under curve measurements were derived from the 10-minute rating task data. Only data from the capsaicin-treated side is shown here. Different letters indicate significant differences (p < 0.05). Error bars represent standard error of the mean values………………………………………………………………….28

Figure 2.2 Group average log irritation intensities of capsaicin (100ppm) over the 10- minute rating period on the tongue, hard palate, cheek, and lip. The “Post 0” points represent the average irritation ratings immediately after capsaicin application on the areas. Error bars represent standard error of the mean values…………………………...30

Figure 2.3 2AFC responses and irritation intensity ratings over time on the tongue (A, B), hard palate (C, D), lip (E, F), and cheek (G, H) are shown here. After initial vehicle and capsaicin (100ppm) stimuli application and ratings (10-minute period), subjects completed a break. After the rest period, capsaicin (100ppm) stimuli were reapplied to each pretreated side and subjects indicated which side felt more irritated and subsequently rated the irritation intensity of each side every 30 seconds. (A, C, E, G) Stars indicate a significant majority (p < 0.05) chose the side not previously exposed to capsaicin (vehicle-pretreated) as feeling more irritated. (B, D, F, H) Stars indicate significantly higher (p < 0.05) irritation ratings on the vehicle-pretreated side compared to the capsaicin-pretreated side. Error bars represent standard error of the mean values..32

Figure 2.4 At the end of the break in between the first and second task, several subjects (9 total) were exposed to the water vehicle on the vehicle- and capsaicin-pretreated sides and indicated which side felt more irritated. Star indicates a significant majority (p < 0.05) chose the capsaicin-pretreated side as feeling more irritated……………………...34

Figure 2.5 (A) Mean max log irritation intensity. (B) Mean area under time intensity curve (AUC). A capsaicin (1000ppm) stimuli was used on the lip and cheek in the same task as described in Figure 1. All other aspects of the figure are the same as in Figure 2.1………………………………………………………………………………………...38

Figure 2.6 Group average log irritation intensities of capsaicin (1000ppm) over time on the lip and cheek. All other aspects of the task and figure are the same as in Figure 2.2………………………………………………………………………………………...39

Figure 2.7 2AFC responses and irritation intensity ratings over time on the lip (A, B) and cheek (C, D) are shown here. All other aspects of the task and figure are the same as in Figure 2.3. ……………………………………….………………………………………40

Figure 3.1 Perceived capsaicin irritation between Caucasian Americans (white bars) and Indians (black bars). (A) Mean max log irritation intensity. (B) Mean area under time intensity curve (AUC). Each mean intensity and AUC displayed represent the mean intensity and AUC across all oral cavity areas. Note, no significant differences in capsaicin sensitivity were found between cultural groups……………………………….58

Figure 3.2 Perceived capsaicin irritation at different oral cavity loci in Caucasian Americans (white circles) and Indians (black squares). (A) Mean max log irritation intensity for the tongue, cheek, hard palate, and lip. (B) Mean area under time intensity curve (AUC) for the tongue, cheek, hard palate, and lip. No significant differences in capsaicin irritation differences were found between Caucasian Americans and Indians at any oral cavity site tested. ………………………………..……………………………...60

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Chapter 1: Literature review

1.1 The Oral Cavity

It is difficult to investigate chemical perception in the mouth without having a thorough understanding of the oral cavity in terms of its structure and neurobiology. In humans, the inner lip eventually joins with the cheek to form the outermost area of the oral cavity. Labial and buccal mucosa line these areas respectively and both connect to the alveolar mucosa which directly attaches to the gingiva, or the gums. Despite their location differences, the labial and buccal lining mucosa are similar due to their lack of a keratinized epithelial layer (Wertz, Swartzendruber, & Squier 1993). Vestibules, or spaces, separate the inner lip and cheek from the lower and upper sets of teeth. The upper set of teeth give way to the arched structure on the roof of the mouth known as the hard palate. Both the hard palate and the nearby gingiva are covered by a tough, keratinized epithelial layer to withstand mastication forces. While the anterior two-thirds roof of the mouth represent the hard palate, the remaining posterior portion is made up of the soft palate, a mix of muscles folded between mucosal lining. Directly below the palate lies the tongue muscle, layered with specialized keratinized and non-keratinized epithelial mucosa. Unique to the tongue are sensory receptors responsible for taste. Aside from the taste sensory pathway, sensory innervation of the oral cavity is provided by the trigeminal nerve (Cohen 2013, Huff & Daly 2019). The tactile receptors, chemoreceptors, and nociceptors that are innervated by the trigeminal nerve respond to various mechanical and chemical stimuli that come in contact with the oral cavity. 1

1.1.1 Tongue, Cheek, Lip, and Hard Palate Mucosa

The tongue is a predominant structure in the oral cavity in terms of its role in swallowing, mastication, taste, and flavor. In humans, the tongue is the primary contributor to sensations of taste. Small dot-like structures known as lingual papillae cover the surface of the tongue. Other than filiform papillae, the foliate, circumvallate, and fungiform papillae all contain taste buds. The receptors connected to these papillae transmit information of the five basic tastes, sweet, sour, salty, bitterness, and umami to the brain. Collectively, the taste or gustatory system is innervated in part by the glossopharyngeal nerve that connects to circumvallate papillae and additionally by the chorda tympani branch of the facial nerve.

Apart from the gustatory system, the trigeminal nerve that encompasses the somatosensory system plays a large role in overall flavor perception on the tongue.

Somatosensory tongue receptors branch from the lingual nerve within the mandibular division of the trigeminal nerve (Figure 1.1) to cause irritating and thermal sensations when activated. The tactile receptors, chemoreceptors, and nociceptors that make up this group of receptors respond to various mechanical stimuli and chemical compounds on the tongue.

Unlike the tongue, the cheek mucosa is not innervated by taste receptors.

However, the mandibular nerve that branches into the long buccal nerve (Figure 1.1) does supply the cheek mucosa with afferent fibers that respond to somatosensory information.

Similarly, afferent nerve fibers that innervate the lower lip mucosa are supplied by the

2 inferior alveolar nerve that also originates from the mandibular branch of the trigeminal nerve. The mandibular branch of the trigeminal nerve is not to be confused with the buccal and mandibular branches of the facial nerve that provide motor innervation to the cheek and lip.

Somatosensory innervation on the hard palate is derived from the maxillary division of the trigeminal nerve. Specifically, the maxillary nerve branches into the nasopalatine and greater palatine nerve to supply the anterior and posterior areas of the hard palate mucosa (Figure 1.2).

Figure 1.1. Branches of the mandibular nerve. The mandibular nerve that originates from the trigeminal nerve innervate the tongue, cheek, and lip mucosa via the lingual, buccal, and inferior alveolar nerve branches, respectively.

Figure 1.2 Branches of the maxillary nerve. The maxillary nerve branches into the greater palatine and nasopalatine nerves to supply the mucosa of the hard palate.

1.2 Capsaicin: Physical and Chemical Properties

Capsaicin, a capsaicinoid chemical compound known for its pungency or spiciness, is a solid compound produced naturally in capsicum peppers. Specifically, the condensation of vanillyamine and fatty acids on the placental walls of capsicum peppers leads to capsaicin synthesis (Reyes-Escogido, Gonzalez-Mondragon, & Vazquez-

Tzompantzi 2011). Dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin are other less commonly found members of the capsaicinoid family. Formally known as 8-methyl-N-vanillyl-6-nonenamide, capsaicin is a non-polar molecule made up of an aromatic ring and amide group with hydrophobic side chain (Fig.

1.3, PubChem). Particularly, the phenol group and length of acyl chain of capsaicin have 5 been found to play an important role in receptor binding of the compound (Katritzky, Xu,

Vakulenko, Wilcox, & Bley, 2003, Barbero et al. 2010). Due to the hydrophobic side chain, capsaicin is insoluble in water but readily soluble in fat, oil, and ethanol.

Figure 1.3 Capsaicin Chemical Structure. Taken from PubChem.

1.3 Neurobiology & Pharmacology of Capsaicin

In humans and animals, capsaicin is a noxious chemical stimulus that has been found to activate transient receptor potential vanilloid 1, a receptor channel associated with sensory neurons (Caterina & Schumacher 1997). Nociception is associated with the

TRPV1 receptor channel, which acts to detect pain and high temperatures. The binding of capsaicin to TRPV1 causes an influx of Calcium ions to enter into the cell. The resulting depolarization causes nerve firings or impulses to be sent from afferent sensory neurons

6 to the brain to signal burning pain (Marsh, Stansfield, Brown, Davey, & McCarthy 1987).

In addition to capsaicin, a variety of other stimuli such as acidic conditions, allyly isothiocyanate, and temperatures above 43C can activate the polymodal TRPV1 channel

(Tominaga et al. 1998).

1.3.1 Desensitization of the Capsaicin Receptor

Through stimulation of single C-polymodal nociceptors, capsaicin has been shown to cause a decrease in nociceptor impulse firings after a previous exposure

(Kenins 1982). Generally, Ca2+ ions released during capsaicin activation have been shown to, in part, cause TRPV1 channel desensitization. Among the desensitization mechanisms hypothesized is dephosphorylation of TRPV1 channels by a

Ca2+/calmodulin-dependent enzyme known as calcineurin. This hypothesis is in contrast to a model presented by Lishko and others that demonstrated potential inhibition of channel activity specifically by Ca2+/calmodulin (2007). However, recurrent capsaicin stimulation may alter desensitization. The complex relationship between

Ca2+/calmodulin-dependent protein kinase 2 (CAMKII) and extracellular signal-regulated kinase (ERK1/2), which are modulated by Ca2+ released after capsaicin stimulation, may play a role in overcoming desensitization after repeated application to neurons (Zhang,

Daugherty, & de Groat 2011).

Along with temporary physiological desensitization, TRPV1 receptors may change as a result of high-level capsaicin exposure. Szallasi and Blumberg first noted vanilloid receptor loss in rats following exposure to a capsaicin analog 24 hours prior

(1992). It has been demonstrated more recently that prolonged exposure of TRPV1 channels to capsaicin or other agonists may lead to endocytosis of sensory receptors.

Endocytosis of the receptors appears to be directly triggered by activation of TRPV1

(Sanz-Salvador et al. 2012).

1.4 Psychophysical Capsaicin Research in the Oral Cavity

In the oral cavity, application of capsaicin results in a characteristic sensation of pain, irritation, burning, or “spiciness” (Rosenbaum & Simon 2007). Capsaicin perception in the oral cavity has been extensively studied by use of psychophysical methods. Application typically involves a whole mouth rinse (Green 1986; Schneider,

Seuß-Baum, & Schlich 2015), though filter papers (Prescott & Swain-Campbell 2000) and cotton swabs (Karrer & Bartoshuk 1990) on the tongue are also commonly used.

Early work by Stevens and Lawless (1987) explored the specificity of chemical receptors in the oral cavity by comparing crossed and same samples of capsaicin and piperine.

When panelists were sequentially exposed to different stimuli, irritation substantially increased at the second exposure compared to exposure to the same stimuli. This suggests recruitment, at least partially, of different chemical receptors between the two stimuli.

Capsaicin has since been studied independently, as a chemical irritant acting on TRPV1 in its own right (Green 1991), but also with other chemical irritants such as citric acid, menthol, and nicotine to explore interactions (Cliff & Green 1996, Dessirier, O’Mahony,

& Carstens 1997, Dessirier 2000). The sensitization and desensitization phenomena

8 involved in capsaicin exposure make it a worthwhile compound to study with major relevance in the food and medical fields.

1.4.1 Sensitization, Desensitization, and Stimulus-Induced Recovery

Sensitization and desensitization due to capsaicin exposure have not only been studied directly with nerve fibers, but on skin surfaces as well. Desensitization to capsaicin was first exhibited on the human tongue by Szolcsányi in 1977 (1977).

Szolcsányi also showed that in a desensitized state, the human tongue had higher thresholds for warm discrimination. Sensitization, along with desensitization, has since been closely examined as well (Green 1989, 1991). When capsaicin is applied successively on the tongue, intensity of irritation steadily increases or sensitizes.

However, application of a longer inter-stimulus interval (ISI) results in a dramatic drop off in irritation intensity or desensitization (Figure 1.4). At a nominal concentration of 3 ppm, the rate of capsaicin sensitization has been shown to be greatest at an ISI of 30 seconds and decrease as the ISI is raised in increments. Though data was inconclusive at a concentration of 3 ppm, an ISI of 10 minutes compared to 15 minutes was shown to be the optimum ISI in regard to desensitization at a concentration of 30 ppm (Green 1991).

This desensitization phenomenon can be chronic and last a period of days (Carstens et al.

2007) or longer following repeated exposure to capsaicin (Karrer & Bartoshuk 1991).

If capsaicin application on the tongue is sustained while in a desensitized state, irritation can return to its original level. This phenomenon, known as stimulus-induced recovery (SIR), has been shown to occur at low and high concentrations and regardless of

9 cumulation of exposures (Green 1996). However, characteristics of capsaicin SIR may differ based on application method and area of exposure. In rats, repeated application induced only partial SIR while a constant flow method demonstrated complete SIR

(Dessirier, Simons, Sudo, Sudo, & Carstens 2000). Green (1998) showed that the time course of SIR on facial skin also progresses differently than lingual skin. On the cheek, most likely due to decreased penetration through mucosal tissue or an epithelial barrier, takes more time to complete SIR compared to the tongue tip.

Sensitization, desensitization, and stimulus-induced recovery are characteristics of capsaicin that have been well supported but remain complex due to the various capsaicin concentrations, mediums, application methods, and areas of application used in previous research.

1.4.2 Modulation by Other Chemesthetic Stimuli

TRPV1 is one receptor among a subfamily of TRP channels that are activated by a variety of chemical irritants. Although TRPV1 activated compounds such as citric acid

and piperine have unsurprisingly been found to cross-desensitize with capsaicin

(Dessirier 2000, Green 1996), other chemesthetic compounds have been shown to have complex relationships with the compound.

Menthol is known to produce coolness and even pain at high enough concentrations. The compound can act to decrease TRPV1 activity and consequently cool the burning pain elicited by capsaicin. Cliff and Green (Cliff and Green 1996)

11 demonstrated asymmetrical desensitization between capsaicin and menthol. Although desensitization to menthol was elicited after capsaicin exposure, capsaicin irritation sensitized after exposure to menthol. Menthol and capsaicin do not likely activate sensory fibers in an identical mechanism of action, but are greatly affected by one another.

Evidence of a relationship was further supported by a study by Kalantzis and others

(2007), where regular compared to non-regular chili-eaters were found to have increased warm detection thresholds and temporary menthol exposure increased thermal thresholds, on average, among the entire group.

Cross-desensitization has also been reported between capsaicin and nicotine

(Dessirier, Simons, Sudo, Sudo, & Carstens 2000), although nicotine has been shown to have different properties regarding sensitization and time-course of desensitization

(Dessirier, O’Mahony, & Carstens 1997, Carstens et al. 2007).

1.4.3 Capsaicin Perception Across Non-Lingual Areas

Although capsaicin receives much attention in studies regarding oral physiology and psychophysics, the tongue or the entire mouth are almost exclusively used to study it. Other areas in the oral cavity are nearly unexplored in capsaicin perception research.

Two studies highlighted perception of capsaicin on areas other than the tongue that are within or near the oral cavity (Lawless, Rozin, & Shenker 1985; Rentmeister-

Bryant & Green 1997). Lawless and Stevens (1988) demonstrated that individually exposed areas in the mouth such as the lip, cheek, hard palate, and various locations on the tongue differ in irritation intensity across a 5-minute period. The tongue tip was found

12 to be the most sensitive location to irritation, followed by the lip and tongue side.

Contrarily, Rentmeister-Bryant and Green (1997) found the throat had the highest sensitivity compared to the posterior and anterior tongue. More surprisingly, irritation between the throat and the posterior hard palate were not found to be substantially different, although it should be noted that a sip-and-swallow method was used. The behavior of other areas within and surrounding the oral cavity in response to capsaicin is still relatively unclear. Although it is evident the tongue is not the only oral cavity structure playing in a role in perception, more research is necessary to investigate capsaicin’s effect on non-lingual oral cavity areas.

1.5 Importance and Use of Chili Pepper Among Cultures

Diet and food choice play a defining role in cultures across the world. For generations, chili peppers have been a popular food ingredient in Central and South

American, Asian, and Middle Eastern original cuisine. In Mexico, as many as 70-80% of the population consume spicy chili peppers regularly (Vera-Guzmán et al. 2017).

Although capsaicin-containing chili peppers evoke painful and seemingly adverse sensations, these high usage levels seen across cultures is indicative of large spicy food acceptance and liking. These preferences have been studied by Rozin in Mexican populations, where there is reported to be a gradual increase in spicy preference during childhood that positively correlates with chili pepper exposure (Rozin & Schiller 1980).

Between Mexican and American groups, there is a lack of support that decreased sensitivity (as measured by thresholds) among Mexican subjects results in increased chili

13 pepper preference (Rozin & Schiller 1980). Although decreased sensitivity may not result in higher preference, high tolerance to chili peppers is often anecdotally observable among cultural groups accustomed to spicy foods.

1.6 Use of Capsaicin as a Model of Chili Pepper Irritancy

Although chili peppers contain a variety of pungent capsaicinoid compounds, the majority of the total capsaicinoid content (71%) is made up by capsaicin (Kosuge &

Furuta 1970). Capsaicin content varies widely across varieties of chili peppers. While mildly pungent bell peppers contain no capsaicin, a study by Thomas et al. (1998) demonstrated that more pungent varieties such as the moderately hot serrano and very hot habanero pepper contain approximately 0.67 and 12 mg capsaicin/g dried pepper, respectively. The capsaicin content demonstrated across these peppers positively drives pungency. This important role of capsaicin in chili pepper pungency has made it a worthwhile compound for use in sensory evaluation studies related to spicy food acceptance and perception. Liking and irritation intensity of food matrices or simple solutions with varying capsaicin concentrations have been previously measured in sensory studies (Prescott 1999; Hernandez & Lawless 1998). The painful, burning sensations experienced from capsaicin exposure allow researchers to mimic the experiences associated with eating chili peppers, while maintaining experimental control.

1.6.1 Capsaicin Sensitivity and Chili Pepper Consumption

In an effort to better understand the eating experience involved in consumption of spicy foods, researchers have investigated how chili pepper consumption is related to capsaicin sensitivity. In a study by Lawless and others (1985), infrequent users were shown to have a dramatically reduced sensitivity to oral capsaicin rinses compared to frequent users. More recent research has shown similar discrepancies, albeit smaller, in capsaicin perception between “high” and “low” chili pepper-intake groups (Nolden &

Hayes 2017). Unsurprisingly, these differences are also found across non-likers and likers of chili pepper given the strong relationship between food choice and liking (Stevenson

& Yeomans 1993). These observational differences in perception have been further supported through experimental studies. In a study by McBurney et al. (1997), subjects were first exposed to capsaicin filter papers on the tongue, sucked on a capsaicin hard candy daily until day 4, and were re-exposed to a capsaicin filter paper on day 5. The dramatic reduction in irritation intensity of the capsaicin stimulus that was demonstrated from day 1 and day 5 support the impact of repeated exposure (or consumption) of capsaicin-containing foods on sensitivity to capsaicin. There is not complete consensus on whether this effect is mainly due to contextual effects or chronic desensitization of capsaicin receptors. Prior ratings of capsaicin stimuli in a laboratory setting could potentially decrease subjects’ subsequent ratings due to changes in reference points

(Stevenson & Prescott 1994), However, a study by Hayes (2000) showed subject groups that both rated irritation of capsaicin stimuli before and after their given treatment

(capsaicin vs. citric acid) still differed in capsaicin sensitivity. Following treatment, the

15 capsaicin-only group were found to have decreased sensitivity compared to the group treated with citric acid in between capsaicin exposures. These findings that include implementation of a control for context provide better support for a physiological change, such as chronic desensitization, between the groups. Although chronic desensitization to capsaicin has been widely studied (Virus & Gebhart 1979; Jessell, Iversen, & Cuello

1978), it still remains difficult to definitively attribute this phenomenon to the demonstrated differences between chili pepper user groups in terms of oral cavity capsaicin sensitivity. Regardless of the mechanism for this phenomenon, there is substantial evidence of the effect that consuming chili peppers has on perceived irritation of capsaicin.

Chapter 2: Assessing regional sensitivity and desensitization to capsaicin among oral cavity mucosae.

2.1 Abstract

This study aimed to determine perceptual response differences and characterize desensitization to capsaicin over time across several oral cavity mucosae– the tongue, cheek, hard palate, and lip. For each region, subjects rated the intensity of capsaicin and a vehicle control over a 10-minute period. Following a rest period, capsaicin was reapplied on each pretreated area and subjects indicated which side felt more irritated then rated each side every 30 seconds, over 3.5 minutes. Results from the initial task indicated significantly greater irritation on the tongue than hard palate, hard palate than cheek and lip, but no significant differences between the cheek and lip. Time to max intensity was delayed on the hard palate compared to the tongue, cheek, and lip. Desensitization, as indicated by a significant proportion of subjects choosing the vehicle-pretreated side over capsaicin-pretreated side as having stronger irritation, was exhibited on the tongue and hard palate, but not the cheek and lip. Given these data, a secondary experiment that utilized a higher capsaicin concentration was conducted on the cheek and lip only.

Results showed significantly higher overall irritation on the lip than the cheek.

Desensitization was exhibited on both areas, although the extent was greater on the lip.

Based on differences in sensitivity and the extent of desensitization among these areas, these results indicate oral cavity mucosae respond to, but are impacted differently by, capsaicin exposure.

2.2 Introduction

Stimuli such as capsaicin, a bioactive compound found in chili peppers, in addition to low pH, ethanol, and heat (>43°C), can cause painful, burning sensations through activation of the receptor TRPV1 (Caterina & Schumacher 1997; Zygmunt et al.;

Trevisani et al. 2002). The TRPV1 receptor also responds to endogenous compounds such as bradykinin and contributes to processing of nociceptive signals (Carr, Kollarik,

Meeker, & Undem 2003). Due to the high expression of TRPV1 in trigeminal neurons

(Tominaga et al. 1998) and the role of capsaicin in pungency of spicy foods, it is unsurprising that oral cavity responses to capsaicin have been well-characterized in psychophysical studies. However, capsaicin psychophysical research has typically been limited to the tongue or generally addressed the oral cavity through whole-mouth rinse protocols. Due to varying physiological or anatomical characteristics including receptor density and distribution or epithelium type that may differentially impact oral mucosal responses to capsaicin, previous research leaves a deficit in knowledge surrounding the regional effects of capsaicin exposure in the oral cavity.

Although a study by Lawless and Stevens (1988) investigated the effects of localized capsaicin treatment across several oral cavity areas, the time-course of irritation was not fully addressed. Over the 5-minute measurement period used, none of the stimulated oral cavity regions had reached peak irritation and begun to diminish in intensity. Given the potential differences in irritation progression among oral cavity areas, extended evaluation time is important to gain a complete understanding of the behavior of oral cavity tissues in response to capsaicin exposure. While Lawless and

Stevens (1988) found the tongue to be most sensitive to capsaicin among several oral cavity areas, a study using whole-mouth rinses (Rentmeister-Bryant & Green 1997) indicated there were no sensitivity differences to capsaicin between the tongue and hard palate. The collection of these results highlight the need for further research clarifying capsaicin sensitivity among oral cavity areas.

After initial exposure to capsaicin, TRPV1 receptors become less responsive to the compound if a rest period is allowed before a subsequent application. This phenomenon, known as desensitization or tachyphylaxis, is largely dependent on the presence of extracellular calcium ions that have flowed into cells after initial exposure to capsaicin ((Liu and Simon 1996); Koplas, Rosenberg, & Oxford 1997). Capsaicin desensitization has also been demonstrated at the cellular level (Dessirier, Simons, Sudo,

Sudo, & Carstens 2000) and in psychophysical studies in the oral cavity (Green 1991).

Although capsaicin has been found to have potential pain-reduction capabilities through this mechanism (Fattori, Hohmann, Rossaneis, Pinho-Ribeiro, & Verri 2016), no study to date has well-characterized desensitization across specific oral cavity areas other than on the tongue.

The overarching goal of these studies was to investigate temporal properties of capsaicin irritation, including desensitization, on specific oral tissues. Such research can provide a better understanding of capsaicin sensitivity across the oral cavity and contribute useful information towards research regarding the use of capsaicin as an analgesic in the oral cavity. Through utilization of a 10-minute intensity rating task and, following reapplication of capsaicin, forced-choice and intensity rating tasks, the specific

19 aims of these studies were 1) determine differences in capsaicin perceptual responses over time among the tongue, cheek, lip, and hard palate mucosae and 2) characterize desensitization to capsaicin over time on each of these areas. It was hypothesized that perceptual responses would differ across the tissues and that, based on the likely presence of TRPV1 receptors across the oral cavity, desensitization would be observable at all areas evaluated.

2.3 Experiment 1

2.3.1 Methods

2.3.1.1 Subjects

Fifty-nine subjects (32 males and 27 females; mean age = 27.4 years; age range =

20-38 years) were recruited from the Columbus, Ohio area to participate in the experiment. All subjects provided written informed consent to participate in protocols that were approved by the Ohio State Institutional Review Board (2013B0277), and guidelines from the Declaration of Helsinki for medical research using human subjects were followed. In an effort to answer an additional research question regarding cultural impact on sensitivity to capsaicin, thirty of the recruited subjects were Caucasian

Americans and twenty-nine were Indians. The results regarding culture are not discussed in the present paper. Potential subjects completed a modified survey questionnaire

(Lawless, Rozin, & Shenker 1985) regarding chili pepper and spicy food use and liking.

To avoid aversion to capsaicin, only subjects that reported consumption of chili pepper-

20 containing foods at least three to four times/week, preferred spice level at least slightly spicy, and liking chili pepper at least slightly were recruited to participate in the study.

Other subject screening criteria included no tobacco use, no tongue, cheek, or lip piercings, no known taste or smell deficits, no history of chronic pain, and no known adverse reactions to capsaicin. Subjects were instructed not to consume any food containing chili pepper or use any products containing menthol for at least 48 hours and 1 hour prior to testing, respectively. These restrictions were placed due to prior research showing desensitization to capsaicin lasting up to 48 hours (Carstens et al. 2007), and potential cross-sensitization to capsaicin by menthol (Cliff and Green 1996).

2.3.1.2 Stimuli

Capsaicin (98%, Enzo Life Sciences, Ann Arbor, MI) at a concentration of 100 ppm in 50% v/v ethanol was used as the stimulus and 50% v/v ethanol was used as the vehicle control. Ethanol was used to solubilize the capsaicin. Prior to testing, 18 µL aliquots of the stimulus (1.8 µg capsaicin) and vehicle were pipetted onto filter papers (1 cm diameter, Whatman, Maidstone, United Kingdom) and allowed to dry completely to ensure evaporation of all ethanol from the filter papers. Any aldehydes and ketones from the ethanol that remained on the filter papers would likely have a negligible effect on irritation perception of capsaicin.

2.3.1.3 Procedure

Data were collected at The Ohio State University (Columbus, OH) in a well-lit, sensory evaluation room designed for psychophysical testing purposes. During test sessions, subjects were seated in a reclining chair, and an overhead light was used by the experimenter to ensure adequate positioning of stimuli on the oral cavity areas. Subjects recorded all responses on Compusense (Guelph, Ontario, Canada) sensory acquisition software on an electronic tablet (Samsung, Seoul, South Korea).

All subjects completed two test sessions at least 48 hours apart due to potential extended effects of desensitization. Each session was split into treatment groups of either the tongue and the cheek, or the hard palate and the lip. These oral cavity areas were grouped (tongue/cheek and hard palate/lip) to reduce subject fatigue and prevent spreading of stimuli on close-contact areas such as the tongue and the hard palate during a session. The session order was randomized and counterbalanced, as was the order of the oral cavity areas within each respective test session.

Irritation intensity measurements were collected using a generalized Labeled

Magnitude Scale (gLMS; Bartoshuk et al. 2004). Prior to data collection, subjects were informed of the nature of this scale and provided with example intensity ratings of sensations such as the brightness of a dim light, room light, and the brightest light possible. As a warm-up exercise, subjects were asked to rate the intensity of remembered and conceptualized sensations including the bitterness of black coffee and the coldness of an ice cube placed on the forearm for five seconds on a provided gLMS.

Each oral cavity area was evaluated individually. The specific application areas were as follows: dorsal surface of the anterior tongue, medial mucosa of the cheek,

22 posterior hard palate adjacent to the midline and medial to the second premolars, and mucosa of the lip rostral and adjacent to the frenulum. During data collection on the lip, subjects were required to use a gloved hand to extend their lip as a means of preventing stimuli contact on the gingiva. However, previous research has demonstrated that when the oral cavity is exposed, evaporative cooling in the oral cavity may decrease intensity of capsaicin (Green 1986). To maintain consistency, subjects were instructed to keep their mouth open during data collection on the other oral cavity treatment areas (tongue, cheek, and hard palate). In an additional effort to avoid stimuli dispersion, subjects were instructed to limit mouth movement during data collection and did not drink water nor talk during the duration of the experiment.

The procedure for each treatment area was as follows. Immediately prior to each application, subjects were instructed to close their eyes and open their mouth or extend their lower lip. A capsaicin- and vehicle-containing filter paper were each soaked with 18

µL of water containing blue or orange dye (Sensient, Milwaukee, WI), respectively.

Forceps were used to simultaneously place the filter papers, randomized to either the left or right side, on the area of interest. Upon application, subjects opened their eyes and immediately rated the irritation intensity on each side and repeated this evaluation every

30 seconds (10 minutes total) thereafter. At the three-minute mark, the filter papers were removed and discarded. Following a mandated rest period of at least 18 minutes and no sensation reported on the stimulated area, subjects moved onto the desensitization task.

Two filter papers, both containing capsaicin, were soaked with 18 µL of blue dye and applied to the previously blue and orange dyed areas that represented the respective

23 capsaicin- and vehicle-pretreated locations. Every 30 seconds (3.5 minutes total), subjects responded to a 2-alternative forced-choice (2-AFC) question regarding which side felt more irritated and subsequently rated the irritation intensity on both sides. Prior to moving on to the next treatment area, subjects rested at least five minutes until their reported irritation intensity on the area fell below a “weak” scale rating and was therefore negligible.

2.3.1.4 Data Analysis

All irritation ratings were log transformed due to the tendency of data collected on with the gLMS to be log-normally distributed (Green et al. 1993). The gLMS is labeled as follows: 0.15 “barely detectable”, 0.78 “weak”, 1.2 “moderate”, 1.5 “strong”, 1.7

“very strong”, 2 “strongest imaginable sensation of any kind”.

Based on the 10-minute rating task, measurements of max intensity, time to max intensity, and area under the time intensity curve (AUC) were extracted from the data for each subject. Max intensity represents the peak intensity experienced, while AUC is a measure of the total sensory response over the time period. Area under the curve measurements were specifically calculated with Microsoft Excel (Microsoft, Redmond,

WA) using the trapezoidal approximation method. A mixed-model ANOVA and

Bonferroni post-hoc tests were performed in SPSS (IBM, Armonk, NY) to determine the effect of sex, frequency of use, and oral cavity area on each these measurements. To determine if there was a relationship between age and max intensity/AUC on each of the oral cavity areas, correlational analyses (two-tailed, α=0.05) were conducted. Average

24 irritation intensities at each time point (30 second intervals) were calculated to build time intensity curves of each oral cavity area (10 minutes total). For some of these specific time points, repeated measures ANOVAs were conducted to determine differences among oral cavity areas. To determine if there was an interaction between area and time, a repeated measures ANOVA test was performed with oral cavity area and time as within-subject factors and subjects as the between-subject factor.

To measure desensitization, data from the 2-AFC tasks were analyzed using the binomial test to determine if a significant proportion of subjects chose the previously- vehicle treated side as more irritating at each time point. A paired t-test was used to compare the corresponding irritation ratings at each time point on the previously-vehicle and -capsaicin treated sides. Both tests were one-tailed (α=0.05) in the direction of the hypothesis (greater proportion and higher rating of previously-vehicle side).

2.3.2 Results

Subject age, sex, and frequency of chili pepper use characteristics can be seen in

Table 2.1. There was a non-significant effect of sex on max intensity and AUC (F1, 48 =

0.552, p = 0.461; F1, 48 = 1.299, p = 0.260, respectively). Max irritation intensity did not significantly differ between males (0.79±0.05) and females (0.87±0.05) and there were no significant differences in AUC between males (318±24.3) and females (341±26.6).

Although there was also no significant effect of frequency of chili pepper use (possible responses ≥3-4 days/week) on max intensity and AUC (F1, 48 = 1.058, p = 0.355; F1, 48 =

1.200, p = 0.310, respectively), there was a significant interaction between sex and

25 frequency of use for AUC (p < 0.05). This interaction indicates that although females displayed decreased AUC (overall sensitivity) with increased frequency of use, AUC of males stayed relatively consistent across frequency of use categories. Correlational analyses of the relationship between age and max intensity/AUC on each of the oral cavity areas only indicated a significant correlation for max irritation intensity on the tongue (r(54) = -0.375, n = 59, p < 0.01).

Table 2.1 Sex, age, chili pepper consumption frequency of subjects in Experiment 1.

Age 3-4 Every day More than

times/wk frequency once a day frequency frequency Males Mean or 27.0 43.9% 40.6% 15.5% (N=32) Percentage SE 0.92 Range 20-38 Females Mean or 27.4 59.2% 36.7% 7.5% (N=27) Percentage SE 0.75 Range 20-38

Total Mean or 27.5 51.5% 38.6% 9.8%

(N=59) Percentage

SE 0.69 Range 20-38

There was a significant effect of oral cavity area on the irritation responses. Max intensity from the 10-minute task significantly differed among areas in the oral cavity

(F3,144 = 56.309, p < 0.001). As displayed in Figure 2.1A, the max intensity on the tongue

(1.29±0.03) was significantly greater than the hard palate (1.00±0.06), the hard palate was greater than the cheek (0.46±0.05) and lip (0.56±0.05) (p’s < 0.001), but there were

26 no significant differences between the cheek and lip (p = 0.532). Similar results (F3,144 =

61.5, p < 0.001) were found with AUC measurements with tongue (589±23.3) significantly greater than hard palate (430±31.9) (p < 0.001), the hard palate greater than the cheek (130±19.5) and lip (169±21.7) (p’s < 0.001), but no significant differences between the latter two tissues (p = 0.887) (Fig. 2.1B). However, this trend was not present at each time point. Initially at time 0, the tongue was significantly more irritated than all other areas (F3,174 = 92.0, p < 0.001). However, once time progressed to 5.5 minutes, irritation intensities on the tongue and hard palate were significantly higher than on the lip and cheek (F3,174 = 86.3, p < 0.001), but the tongue and hard palate no longer significantly differed from each other.

In addition to irritation intensity differences among the areas, the shape of the time intensity curve on the hard palate differed dramatically from all other areas. The tongue, cheek, and lip were quick to peak in irritation intensity. In comparison, irritation intensity on the hard palate reached a peak far later than all other areas and appeared to plateau after reaching peak intensity compared to the tongue. Once the hard palate began to decline, it did so at a similar rate to the tongue (Fig. 2.2). Results from a mixed-model

ANOVA test on the time to reach max intensity over the 10-minute period (F3,165 = 18.3, p < 0.001) revealed the time to reach max intensity on the hard palate (4.45±0.31 mins) was significantly greater than on the tongue (2.47±0.22 mins), cheek (2.39±0.28 mins), and lip (2.18±0.28 mins) (p’s < 0.001), but not significantly different among the latter three oral cavity areas (p’s > 0.05). Repeated-measures ANOVA results with area and time as factors showed an area*time interaction effect (F60, 34800 = 24.5, p < 0.001) that supported these differences in irritation progression on the hard palate compared to on the tongue, lip, and cheek.

As expected for the tongue, results from the second capsaicin application indicated that a significant majority of subjects chose the previously vehicle-treated side as feeling more irritated (Fig. 2.3A), and irritation ratings were significantly higher on the vehicle-pretreated side compared to the capsaicin-pretreated side at all time points (Fig.

2.3B). Comparatively, the proportion choosing the vehicle-pretreated side on the hard palate at 0.5 minutes was not significant. After 0.5 minutes on the hard palate, a significant majority chose the vehicle-pretreated side as more irritated, albeit a lower proportion choosing this side compared to on the tongue (Fig. 2.3C; Fig. 2.3A). Irritation ratings on the vehicle-pretreated side were significantly greater than the capsaicin- pretreated side at all time points after 0.5 minutes on the hard palate, which aligned with the corresponding 2-AFC responses (Fig. 2.3D). Compared to the tongue and hard palate,

30 the proportion choosing the vehicle-pretreated side versus the capsaicin-pretreated side as more irritating did not significantly differ at any time points on the lip or cheek (Fig.

2.3E; Fig. 2.3G). However, the intensity ratings did not entirely match 2-AFC responses on these areas, as there were still time points on the lip and cheek at which the ratings were slightly, but significantly greater on the vehicle-pretreated side than the capsaicin- pretreated side (Fig. 2.3F; Fig. 2.3H).

Figure 2.3. 2AFC responses and irritation intensity ratings over time on the tongue (A, B), hard palate (C, D), lip (E, F), and cheek (G, H) are shown here. After initial vehicle and capsaicin (100ppm) stimuli application and ratings (10-minute period), subjects completed a break. After the rest period, capsaicin (100ppm) stimuli were reapplied to each pretreated side and subjects indicated which side felt more irritated and subsequently rated the irritation intensity of each side every 30 seconds. (A, C, E, G) Stars indicate a significant majority (p < 0.05) chose the side not previously exposed to capsaicin (vehicle-pretreated) as feeling more irritated. (B, D, F, H) Stars indicate significantly higher (p < 0.05) irritation ratings on the vehicle-pretreated side compared to the capsaicin-pretreated side. Error bars represent standard error of the mean values.

The absence of desensitization on the lip and cheek and lack of differentiation between the areas was counter to the original hypotheses. However, it should be noted that peak intensity over the 10-minute rating task did not surpass “weak” on the lip, nor the cheek (Fig. 2.1A). It is possible at these faint intensities, the resolution of response differences between the cheek and lip was lower, and perceiving desensitization on the areas was more difficult for subjects. A previous study has also shown decreases in the extent of desensitization at lower intensities on the tongue (Green 1991).

To better understand the desensitization results, a task was conducted to determine subject responses when there was no difference in sensation between treated sides of an area. At the end of the break between the perceptual and desensitization tasks, 9 subjects

(who confirmed they felt no sensation on either side of the treated area) were exposed to vehicle stimuli on the vehicle-pretreated and capsaicin-pretreated locations. After 30 seconds, subjects were asked to choose the side with stronger irritation in the same 2AFC paradigm previously used. Although only the vehicle had been applied, the results showed that a significant proportion chose the capsaicin-pretreated side as more intense

(Fig. 2.4). These findings suggest there was a demand to choose the capsaicin-pretreated side as more intense when there was no way to discern irritation between the sides. This helps to explain the results found from the desensitization task. When intensities from stimuli were lower and likely more difficult to discern for some subjects, there may have been a bias to choose the side previously treated with capsaicin.

Figure 2.4. Following the break between the first and second task, several subjects (9 total) were exposed to the water vehicle on the vehicle- and capsaicin-pretreated sides and indicated which side felt more irritated. Star indicates a significant majority (p < 0.05) chose the capsaicin-pretreated side as feeling more irritated.

Given that the cheek and lip desensitization results may have been partly due to this psychological phenomenon, a follow up study was conducted on the cheek and lip to further investigate perceptual responses differences and elicit desensitization on the lip and cheek. This experiment utilized a similar design but used a higher concentration of capsaicin.

2.4 Experiment 2

The results from Experiment 1 showed differing responses to capsaicin among all areas except the lip and the cheek. Furthermore, desensitization was not clearly observable on the lip and the cheek. It was theorized that the lack of an observed effect

35 may have been a result of the low intensity of capsaicin on these areas. An experiment using capsaicin, at a concentration ten times greater than the concentration utilized in

Experiment 1, was conducted on the lip and the cheek to determine sensitivity differences between the areas and if desensitization could be observed.

2.4.1 Methods

2.4.1.1 Subjects

Seventeen subjects from the Experiment 1 sample participated in the experiment.

As in Experiment 1, subjects were instructed not to consume any food containing chili pepper or use any products containing menthol for at least 48 hours and 1 hour prior to testing, respectively.

2.4.1.2 Stimuli

Capsaicin in 100% ethanol, at a concentration of 1000ppm, was used as the stimulus, and 100% ethanol was used as the vehicle control. The stimulus (18 µg) and vehicle were prepared, wetted, and delivered using the same methods from Experiment 1.

2.4.1.3 Procedure

The lip and cheek areas, in the same locations described in Experiment 1, were the areas measured in this experiment. Due to increased salivation from the higher- concentration capsaicin stimulus, a vacuum suction pump with a saliva ejector attachment was used to remove excess saliva every two minutes during data collection. Breaks were

36 extended to at least 30 minutes between the 10-minute rating task and 2-AFC tasks and at least 15 minutes between areas. All other aspects of the protocol were the same as in

Experiment 1.

2.4.1.4 Data Analysis

Paired, two-tailed t-tests were conducted on max intensity, time to max intensity, and AUC measurements between the lip and cheek. Tests at p < 0.05 were considered significant.

Data from 2-AFC and subsequent intensity rating tasks were analyzed according to methods described in Experiment 1.

2.4.2 Results

Results from paired t-tests showed that max intensity and AUC were significantly

(p = 0.002; p = 0.004, respectively) higher on the lip (1.54±0.05; 776±32.5, respectively) than the cheek (1.18±0.10; 542±58.2, respectively) (Fig. 2.5). There was no significant difference in the time to max intensity between the areas (lip: 4.12±0.41 mins; cheek:

3.35±0.28 mins; p = 0.076).

Figure 2.6 also shows that irritation intensity over the 10-minute period was generally higher on the lip compared to the cheek. 38

Figure 2.6 Group average log irritation intensities of capsaicin (1000ppm) over time on the lip and cheek. All other aspects of the task and figure are the same as in Figure 2.2.

Results from the 2-AFC and irritation intensity rating tasks indicated that, at every time point on the lip, a significant majority (Fig. 2.7A) chose the vehicle-pretreated side as more irritated, and intensity ratings on the vehicle-pretreated side were significantly greater than on the capsaicin-pretreated side (Fig. 2.7B). On the cheek, a significant majority (Fig. 2.7C) chose the vehicle-pretreated side as more irritated at all time points other than 0.5, 1, and 2 minutes. Compared to the lip, the proportion that chose the vehicle-pretreated side on the cheek as more irritating was lower across all time points

(Fig. 2.7A; Fig. 2.7C). Intensity ratings on the vehicle-pretreated side were significantly greater than ratings on the capsaicin-pretreated side at all time points after 1 minute (Fig.

2.7D).

Figure 2.7 2AFC responses and irritation intensity ratings over time on the lip (A, B) and cheek (C, D) are shown here. All other aspects of the task and figure are the same as in Figure 2.3.

2.5 Discussion

These studies investigated regional effects of capsaicin in the oral cavity by assessing sensitivity and desensitization to capsaicin across oral cavity areas. The aim was to reexamine response differences to capsaicin and to characterize desensitization among the tongue, cheek, hard palate, and lip mucosae.

Similar to findings by Lawless and Stevens (1988), Experiment 1 results indicated that the tongue appeared to be the most sensitive region overall, which likely suggests higher receptor density on the tongue compared to the hard palate, lip, and cheek. These results are supported by a prior immunohistochemistry study that demonstrated high TRPV1 innervation on the tongue (Kido, Muroya, Yamaza, Terada, &

Tanaka 2003). Responses on the tongue were also immediate—at the time point after application, irritation on the tongue was greater than on all other areas. In addition to its presence in tongue peripheral nerves (González-Ramírez, Chen, Liedtke, Morales-Lázaro

2017), the TRPV1 capsaicin receptor has also been reported in tongue epithelial cell tissue (Kawashima, Imura, & Sato 2012). A relatively small epithelial barrier to prevent access of capsaicin to TRPV1, in addition to the distribution of TRPV1 on the tongue surface, may contribute to the immediacy of irritation on the tongue in this study.

Irritation on the hard palate, comparatively, progressed much later and took longer than all other areas to reach max intensity in Experiment 1. Unlike the tongue, cheek, and lip mucosae, the hard palate is completely covered by a layer of keratinized epithelial tissue that reduces permeability (Lesch, Squier, Cruchley, Williams, & Speight 1989). This tissue type may have served as an initial barrier to TRPV1 receptors that consequently delayed the response on the hard palate.

Despite the irritation delay, the hard palate was shown to be more sensitive to capsaicin than the cheek, contrary to previous findings (Lawless & Stevens 1988). One explanation for this difference may simply be related to the duration of intensity measurements. The 5-minute measurement period used by Lawless and Stevens (1988)

41 may not have been long enough to observe the overall greater sensitivity on hard palate compared to the cheek that occurred over the 10-minute measurement period in the present study. However, this theory does not explain how, in the current study, the hard palate was higher in irritation intensity than the cheek at most time intervals leading up to

5 minutes. The discrepancies in these findings may also be a product of stimulus placement. In the present study, stimuli were placed on the hard palate in a location medial to the second premolars rather than the canine teeth. The greater responses demonstrated here on the hard palate may be indicative of an increasing gradient of sensitivity from the posterior to anterior palate. The greater sensitivity of the soft palate compared to hard palate that was demonstrated by Lawless and Stevens (1988) also support this theory. Although spreading of solution from the hard to soft palate in the present study cannot be completely ruled out, relatively low salivary flow on the hard palate (Lee, Lee, Chung, Kim, & Kho 2002) and the small filter paper size used would likely mitigate these occurrences. Capsaicin stimuli were also randomized to either side of the hard palate midline in this study, rather than applied directly atop it, as in the study by Lawless and Stevens (1988). Recent research has demonstrated that taste perception may not be equal even across the tongue structure (Higgins & Hayes 2019). It is plausible that capsaicin sensitivity may also vary across the hard palate due to differences in tissue structure, such as the presence of a palatine raphe, receptor density, or salivary flow to the area, resulting in the overall higher sensitivity on the hard palate that was observed in this study. Regardless of the basis for this discrepancy, these findings suggest that certain

42 areas of the hard palate may have greater sensitivity to capsaicin than has been previously shown.

Sensitivity on the cheek and the lip, however, was low and did not differ between the areas in Experiment 1. It may be possible that, due to potentially lower receptor density on the cheek and lip compared to the hard palate and tongue, the slight irritation subjects experienced on the lip and cheek may have been difficult to accurately identify and therefore differentiate. Cheek and lip stimulation at a higher capsaicin concentration in Experiment 2 provided better resolution of differences between the two areas. The results indicated much greater irritation responses on the lip than on the cheek, in correspondence with prior reports of greater somatosensory sensation on the lip compared to cheek mucosa (Lawless & Stevens 1988; Svensson, Bjerring, Arendt-

Nielsen, & Kaaber 1993). Although tissue structure is similar between these areas

(Winning & Townsend 2000) it is plausible that greater TRPV1 receptor density may be responsible for the increased sensitivity observed on the lip compared to the cheek.

Based on results from the 2-AFC and rating tasks in Experiment 1, desensitization was clearly observed on the tongue, which is in agreement with prior studies (Karrer &

Bartoshuk 1991; Green 1991). On the hard palate, cheek, and lip, however, the extent of perceivable desensitization varied at the low concentration. The delayed desensitization observed on the hard palate area provides further indication that the surface epithelial tissue may act as a barrier that initially slows capsaicin stimuli access to TRPV1 receptors. Responses on hairy cheek skin, whose epidermis is also comprised of keratinized epithelial cells, demonstrated similar delayed desensitization in a study by

Green (1998). Notably, the cheek and lip mucosae did not appear to be desensitized to capsaicin in Experiment 1. Although tongue desensitization has been observed using very low concentrations of capsaicin (Green 1989), results from this study show that desensitization may only be perceivable above moderate concentration thresholds on the cheek and lip. As was the case in results from the initial rating task, this can presumably be attributed to lower capsaicin sensitivity on these areas compared to the tongue and hard palate. It is likely that, in the case of the cheek and lip, few receptors were activated and consequently produced low levels of irritation upon initial capsaicin application.

Those few desensitized receptors during reapplication of capsaicin would be unlikely to produce a powerful perceived desensitization effect. Therefore, it was theorized that the low irritation produced on each side during capsaicin reapplication made it too difficult for subjects to differentiate between sides and therefore perceive desensitization on the cheek and lip areas. The desensitization effects that were observed on the cheek and lip at a greater concentration in Experiment 2 support this theory. However, even at a higher concentration of capsaicin, desensitization was not consistently realized on the cheek.

Although the small sample size in Experiment 2 may have contributed to this lack of consistent desensitization, the results still serve as an indication of the low capsaicin sensitivity on the cheek compared to the lip, where desensitization was fully realized at the same sample size and stimulus concentration.

This study was designed to measure the effect of capsaicin exposure on each area in an independent manner. However, the hard palate/lip and cheek/tongue session groups in Experiment 1 may have led to unbalanced context effects on irritation ratings. In

44 previous studies investigating regional oral sensitivity to stimuli, different areas have been measured in one total session (Green 1996) or in individual sessions (Lawless &

Stevens 1988). Due to the potential for fatigue and high likelihood of cross-exposure to capsaicin between areas such as the tongue and hard palate, the former method was not ideal. Individual sessions per area would have likely resolved these issues but did not appear to be feasible due to anticipated difficulty of subject retainment. Therefore, the two different groups of oral cavity areas were a compromise that alleviated cross- exposure to capsaicin among oral areas and recruitment difficulties. Although cross- exposure problems were improved with these groupings, they were not eliminated.

Despite instructions, it is still possible there was contact between areas such as the tongue and cheek that could lead to cross-exposure to capsaicin. However, these effects were likely to be minor as the side of the tongue, rather than the dorsal tongue that was stimulated, would have been most likely to touch the cheek.

The data here serve as an examination of the temporal behavior of different oral cavity areas in response to capsaicin stimuli. Although the tongue, cheek, hard palate, and lip all responded to capsaicin, the nature of those responses and extent of desensitization differed greatly across these areas. Nuanced and large differences alike in receptor distribution and density and tissue structure across the areas likely play a role in the differing capsaicin sensitivity observed. Since capsaicin may be a potential analgesic for afflictions such as oral mucositis and burning mouth syndrome (Berger et al. 1995;

Silvestre, Silvestre-Rangil, Tamarit-Santafé, & Bautista 2012), this study provides necessary sensitivity comparisons and desensitization characterization in the oral cavity

45 to inform further research on its use in medical applications. The varying time course of irritation across these tissues may also impact perception and acceptance of foods containing capsaicin. Understanding and considering these heterogenous temporal responses to capsaicin among various regions in the oral cavity may provide a more comprehensive understanding of the spicy flavor experience.

Chapter 3: Determination of capsaicin sensitivity differences between Caucasian American and Indian cultural groups: A study designed to control for chili pepper affinity.

3.1 Abstract

Although different cultural groups are known to vary in their tolerance for hot chili peppers, the impact of factors such as cultural background and upbringing on sensitivity to spicy food is unclear. A study was designed to investigate sensitivity differences to capsaicin between Caucasian American and Indian cultural groups while controlling for general chili pepper affinity. The two cultural groups were selected to match on metrics related to chili pepper use and liking. Subjects were exposed to a capsaicin (100ppm) stimulus on the tongue, cheek, hard palate, and lip and rated the intensity of irritation every 30 seconds, over a 10-minute period. Overall sensitivity to capsaicin in the oral cavity did not differ between the groups, nor were responses different between the groups depending on the oral cavity area stimulated. These data suggest a limited role of cultural attributes on capsaicin sensitivity between Caucasian Americans and Indians. The methods and findings here provide subject recruitment insight and guidance on effectively designing a sensory study to answer perceptual questions regarding specific subject groups.

3.2 Introduction 47

Across cultural cuisines, the prevalence of ingredients such as pungent chili peppers can vary greatly. The use of chili peppers in food is especially frequent in countries such as India, where chili is the most commonly consumed spice (Siruguri &

Bhat, 2015), and Mexico, where fresh chili consumption is reported to be as high as 20 g chili pepper per day (López-Carnllo et al. 1994). Comparatively, the average American has been reported to prefer only ~1 g chili pepper per spicy meal (Ludy & Mattes, 2011).

Frequency of chili pepper consumption has been primarily investigated as a means of explaining tolerance differences to spicy foods that are often observed across cultural groups. In prior studies where American subjects were split into groups of varying chili pepper consumption frequencies, chili pepper “users” demonstrated lower sensitivity to capsaicin (the main pungent compound in chili peppers) than chili pepper “nonusers”

(Lawless, Rozin, & Shenker, 1985; Ludy & Mattes, 2012). Although these studies strongly support the role of consumption frequency of chili peppers in perception of spicy foods, they fail to thoroughly answer whether ethnic background impacts spicy perception.

Across cultural groups where the ubiquity of spicy cuisine varies, there may be contextual-based differences in capsaicin perception that do not depend on consumption frequency of chili pepper use. A cultural group accustomed to chili peppers in their cultural cuisine may have a decreased perception of spiciness compared to a culture where use of chili peppers is generally uncommon. Physiological differences may also impact capsaicin perception among cultural groups. In a study by Sanz-Salvador et al.

(2012), prolonged exposure to capsaicin in rats was shown to lead to a long-term

48 decreased capsaicin response via the downregulation of capsaicin receptors. Similar findings have been observed in humans where even a single exposure to capsaicin can lead to desensitization lasting days (Karrer & Bartoshuk, 1991; Carstens, Albin, Simons,

& Carstens, 2007). It is possible that duration of consumption of chili peppers, which likely varies across cultures, could also promote receptor expression changes in humans that would cause variation in spicy perception. Differences in initial age of chili pepper consumption was cited by Ludy et al. (2012) as a potential explanation for demonstrated sensory perceptual differences between chili pepper “users” and “nonusers”, but the results were ultimately confounded by the main grouping variable– frequency of use.

Although it is important to mention that cultural groups may also differ in sensitivity due to genetic variation of capsaicin receptors, these aspects are inextricable from cultural differences without conducting genomic analyses. In addition, there is a lack of substantiated evidence attributing individual subject variation in capsaicin perception (Cliff & Green, 1996) to capsaicin receptor differences. Therefore, the main focus of the present study was to investigate the impact of previously unexplored cultural attributes on capsaicin perception. Understanding perceptual differences based on cultural attributes has relevance for balancing ethnicity demographics when recruiting for sensory testing. Furthermore, the study design used here provides a model for answering specific research questions in sensory test settings regarding subject characteristics.

In the present study, potential sensitivity differences to capsaicin were investigated between two cultural groups (Caucasian Americans and Indians) that were matched in terms of frequency of chili pepper consumption and additional measurements

49 of chili pepper affinity. It was hypothesized that the Indian group would demonstrate decreased sensitivity to capsaicin compared to the Caucasian American group after controlling for chili pepper affinity due to differences in cultural background.

3.3 Methods

3.3.1 Subjects

Fifty-nine subjects (32 males, 27 females; 30 Caucasian Americans, 29 Indians) from the Columbus, Ohio area were recruited through flyers and email and provided their written consent to be a part of the study. A sample size of approximately 30 per sample group was determined based on an a priori power analysis, in addition to guidance from prior studies that have evaluated the impact of culture (Bertino & Chan, 1986) and frequency of chili pepper use (Stevenson & Yeomans, 1993) on taste and flavor perception. Study protocols followed the guidelines from the Declaration of Helsinki for medical research using human subjects and were approved by the Ohio State Institutional

Review Board (2013B0277). To emphasize cultural differences between each group, only subjects who had lived in their respective country (United States for Caucasian

Americans; India for Indians) at least 50% of their life qualified for the study. Subjects were also screened to ensure no tobacco use, no tongue, cheek, or lip piercings, no known taste or smell deficits, no history of chronic pain, and no known adverse reactions to capsaicin. Subjects did not consume food or use products containing menthol for 1 hour prior to testing nor did they consume any food containing capsaicin for 48 hours prior to testing. These restrictions were placed due to prior research showing potential cross-

50 sensitization to capsaicin by menthol (Cliff et al., 1996), and desensitization to capsaicin lasting up to 48 hours (Carstens et al., 2007).

3.3.2 Survey Questionnaire Related to Chili Pepper Use and Liking

Prior to selection for the study, potential subjects were required to complete an online modified survey questionnaire (Lawless, Rozin, & Shenker 1985) using Qualtrics

(Provo, UT) that was related to their chili pepper use and liking. A chili pepper use and liking score (CPULS) for each potential subject was calculated based on the sum of the number-coded responses for each survey item (CPULS min = 18; max = 41). The included survey items were as follows: frequency of chili pepper use, preferred spice level, taste and burn liking of chili pepper, and several true/false statements related to spicy food liking (see Appendix B). Only potential subjects that reported consuming foods containing chili peppers at least 3-4 times per week, preferring a spice level of at least slightly spicy, and at least slightly liking the taste of chili pepper qualified to ensure capsaicin could be tolerated. Due to prior research that has demonstrated sensitivity differences between frequent and infrequent users of chili (Lawless, Rozin, & Shenker

1985), it was important to control for chili pepper consumption frequency in the current study. Therefore, the Caucasian American and Indian subject groups were primarily matched on their reported frequency of chili pepper consumption. Furthermore, a similar

CPULS range and mean CPULS was ensured between each cultural group to control for other relevant metrics related to affinity for chili pepper. An additional question regarding the age of initial chili pepper consumption was asked during the survey. This

51 metric was not matched between groups in order to evaluate if duration of prior chili pepper experience impacted perceptual differences.

3.3.3 Stimuli

Capsaicin (98%, Enzo Life Sciences, Ann Arbor, MI) was dissolved in 50% v/v ethanol to form a weak-moderately pungent, 100 ppm concentration solution. Aliquots of

18 µL of the capsaicin solution (1.8 µg of capsaicin) and 50% ethanol were delivered onto filter papers (1 cm diameter, Whatman, Maidstone, United Kingdom) to serve as the stimulus and the vehicle control, respectively.

3.3.4 Procedure

The tongue, cheek, hard palate, and lip mucosa were treated with stimuli in this study. This design allowed a more complete examination of oral cavity sensitivity to capsaicin among the cultural groups. These treatment areas were also chosen in order to answer an additional research question regarding differences in sensitivity to capsaicin among various oral cavity areas. Findings regarding this research question will not be discussed in depth in the present study.

52 recorded all responses on Compusense (Guelph, Ontario, Canada) sensory acquisition software on an electronic tablet (Samsung, Seoul, South Korea).

All subjects completed two test sessions at least 48 hours apart due to potential extended effects of capsaicin desensitization (Carstens et al., 2007). Each session was split into treatment groups of either the tongue and the cheek, or the hard palate and the lip. These oral cavity areas were grouped (tongue/cheek and hard palate/lip) to reduce subject fatigue and prevent spreading of stimuli on close-contact areas such as the tongue and the hard palate during a session. The session order was randomized and counterbalanced, as was the order of the oral cavity areas within each respective test session.

Irritation intensity measurements were collected using a generalized Labeled

Magnitude Scale (gLMS; Bartoshuk et al., 2004). Prior to data collection, subjects were informed of the nature of this scale and provided with example intensity ratings of sensations such as the brightness of a dim light, room light, and the brightest light possible. As a warm-up exercise, subjects were asked to rate the intensity of remembered and conceptualized sensations including the bitterness of black coffee and the coldness of an ice cube placed on the forearm for five seconds on a provided gLMS.

Each oral cavity area was evaluated individually. The specific application areas were as follows: dorsal surface of the anterior tongue, medial mucosa of the cheek, posterior hard palate adjacent to the midline and medial to the canine teeth, and mucosa of the lip rostral and adjacent to the frenulum. During data collection on the lip, subjects were required to use a gloved hand to extend their lip as a means of preventing stimuli

53 contact on the gingiva. However, previous research has demonstrated that when the oral cavity is exposed, evaporative cooling in the oral cavity may impact perceived intensity of capsaicin (Green, 1986). To maintain consistency, subjects were instructed to keep their mouth open during data collection on the other oral cavity treatment areas (tongue, cheek, and hard palate). In an additional effort to avoid stimuli dispersion, subjects were instructed to limit mouth movement during data collection and did not drink water nor talk during the duration of the experiment.

µL of water containing blue or orange dye (Sensient, Milwaukee, WI), respectively.

30 seconds (10 minutes total) thereafter. At the three-minute mark, the filter papers were removed and discarded. Following a mandated rest period of 18 minutes and verbal confirmation that subjects felt no sensation on the stimulated area, subjects moved onto a subsequent task for the same area. Although all subjects completed this second task, the results pertained to a research objective unrelated to the present study and will not be discussed here. Prior to moving on to the next treatment area, subjects rested for five minutes and were required to verbally confirm that the sensation they felt fell below a

“weak” scale rating and was therefore negligible.

2.3.5 Data Analysis

Two-tailed, unpaired t-tests (α=0.05) were conducted to validate that there was no significant difference in age and CPULS between cultural groups. To additionally validate that there was no significant relationship between cultural group and reported consumption frequency of foods containing chili pepper, the Chi-Square test of independence (α=0.05) was conducted. The relationship between cultural group and duration of chili pepper consumption was also investigated using the Chi-Square test of independence (α=0.05).

All irritation ratings were log transformed due to the tendency of data collected on with the gLMS to be log-normally distributed (Green, Shaffer, & Gilmore, 1993). The gLMS is labeled as follows: 0.15 “barely detectable”, 0.78 “weak”, 1.2 “moderate”, 1.5

“strong”, 1.7 “very strong”, 2 “strongest imaginable sensation of any kind”. Area under the curve (AUC), which was calculated using a trapezoidal approximation method in

Microsoft Excel (Microsoft, Redmond, WA), and max irritation intensity were derived from the 10-minute rating task for every subject on each oral cavity area. A mixed-model

ANOVA test was conducted using culture, sex, and frequency of use as between-subjects factors and oral cavity area as within subject factors. Although the primary research question regarded culture, sex and frequency of use were evaluated to control for potential confounds, and oral cavity area allowed for evaluation of potential interactions between culture and oral cavity area.

3.4 Results

Subject characteristics are displayed in Table 3.1. There were no significant differences between the average age of the Caucasian American group (mean: 27.5, range: 20-39) and that of the Indian group (mean: 27.4, range: 20-38) (p = 0.98). The

Chi-Square test on chili pepper consumption duration revealed Indians were significantly more likely to have consumed chili peppers since childhood compared to Caucasian

Americans, χ2 (2, N = 59) = 8.93, p < .05. The reported consumption of foods containing chili pepper did not differ by cultural group, validating that cultural groups were matched on this characteristic, χ2 (2, N = 59) = 0.8, p > .05. The average CPULS in each group was also effectively matched, as there was no significant difference in CPULS between

Caucasians (mean: 34.6, range: 28-40) and Indians (mean 34.7, range: 26-40) (p = 0.88).

Table 3.1. Sex, age, chili pepper consumption frequency, age began consuming chili peppers, and CPULS between Caucasian American and Indian subject groups. Caucasians (N = 30) Indians (N = 29)

Sex Males 15 17

Females 15 12

Age 27.5 27.4

Consumption of 3-4/wk frequency 53.3% 47.5% foods containing chili pepper Every day frequency 40.0% 36.0%

More than once a day 6.7% 16.5%

frequency

Age began Since childhood 60.0% 92.6% consuming chili pepper ** Since young adulthood 40.0% 7.4%

CPULS 34.6 34.7

** chi-square test is significant at p < 0.01.

Results from ANOVA tests showed no significant effect of culture on max irritation intensity (F1, 48 = 0.838, p = 0.365) or AUC (F1, 48= 1.336, p = 0.253) from the

10-minute task. Specifically, it was revealed that there was no significant difference in max irritation intensity between Caucasian Americans (0.83±0.05) and Indians

(0.82±0.05), nor was there a significant difference for AUC between Caucasian

Americans (334±25.0) and Indians (325±25.9) (Fig. 3.1). Similarly, there was a non-

significant effect of sex on max intensity (F1, 48 = 0.552, p = 0.461) and AUC (F1, 48 =

1.299, p = 0.260) and of frequency of use on max intensity (F1, 48 = 1.058, p = 0.355) and

AUC (F1, 48 = 1.200, p = 0.310).

There was a significant effect of oral cavity area on max irritation intensity (F3,144

= 56.309, p < 0.001) and AUC (F3,144 = 61.5, p < 0.001) from the 10-minute task.

However, the oral cavity area*culture interaction was not significant for max intensity

(F3, 144 = 0.084, p = 0.969), nor AUC (F3, 144 = 0.108, p = 0.955), showing that AUC and max intensity responses between cultural groups did not depend on the oral cavity area that was stimulated (Fig. 3.2).

Figure 3.2. Perceived capsaicin irritation at different oral cavity loci in Caucasian Americans (white circles) and Indians (black squares). (A) Mean max log irritation intensity for the tongue, cheek, hard palate, and lip. (B) Mean area under time intensity curve (AUC) for the tongue, cheek, hard palate, and lip. No significant differences in capsaicin irritation differences were found between Caucasian Americans and Indians at any oral cavity site tested.

3.5 Discussion

The careful control measures implemented in the present study allowed us to determine if, aside from factors related to chili pepper affinity, two cultural groups 60 differed in their sensitivity to capsaicin. Caucasian Americans and Indians were matched on chili pepper use and liking metrics but differed in cultural background. Although the hypothesis that Indians would demonstrate lower sensitivity to capsaicin compared to

Caucasian Americans was not supported by the findings, the original objective question was answered effectively. Between American Caucasian and Indian cultural groups, oral cavity sensitivity to capsaicin was found to be strikingly similar and did not differ based on which oral cavity area was stimulated. Contrary to the present findings, prior studies where capsaicin was used to induce trigeminal sensitization have reported pain sensitivity differences across cultural groups and sex (Gazerani, Andersen, & Arendt-Nielsen, 2005;

Gazerani & Arendt-Nielsen, 2005). However, differences in the capsaicin stimulation route (orally vs. intradermally) and degree of sensation (irritation vs. pain) may be responsible for the discrepancies between these results.

The findings here should not be interpreted as an overall lack of difference in oral capsaicin perception between Caucasian American and Indian cultural groups. A prior study showed capsaicin sensitivity differences across groups that differed in terms of their spicy food use, liking, and age of introduction (Ludy et al., 2012). In the present study, consumption frequency and liking of chili peppers were purposefully matched, but more Indians reported consuming chili pepper since childhood compared to Caucasian

Americans. Despite this, no sensitivity differences were found between the groups. The combination of these findings may suggest that cultural attributes, including duration of prior chili pepper experience, play a minimal role in capsaicin sensitivity compared to other factors such as consumption frequency or liking of chili peppers. Therefore,

61 capsaicin sensitivity differences may still exist between Caucasian Americans and

Indians due to the differing diets between the groups.

The design of this study illuminated the lacking effect of cultural background on perceived intensity of capsaicin between Indians and Caucasian American groups. It is of value to know how perception may or may not differ across demographics such as ethnicity and sex when recruiting panelists for sensory evaluation tests. The lack of perceived differences of capsaicin found in this study may suggest that, when recruiting for sensory tests that include spicy flavored products, ethnicity and sex demographics may not be important factors to consider if consumption habits have already been screened for. This knowledge may alleviate pressure sensory scientists often face to recruit diverse samples for tests. However, future tests across a variety of cultural groups would be necessary to confirm this hypothesis.

This study design also provides a model for researchers interested in utilizing sensory testing to answer questions about subject groups. While the objective of this study regarded sensitivity differences across cultural groups, alterations in the subject matching process used presently could be easily implemented to investigate sensitivity across other differing subject characteristics of interest. Other measurements, such as liking and preference, are also able to be used in a generalized version of this study model. The benefit of such a design is that confounding variables are minimized effectively, providing researchers with higher confidence in study findings.

Chapter 4: Overall conclusions

This study explored capsaicin irritation and desensitization throughout the oral cavity. The tongue, cheek, lip, and hard palate were found to be differentiated in terms of perceptual responses to capsaicin. Although previous findings have demonstrated response differences to chemesthetic stimuli across oral areas (Lawless & Stevens, 1988), this study provided novel data on progression differences and greater hard palate sensitivity than previously found. In addition to understanding sensitivity differences, desensitization was characterized on each of the oral cavity mucosae. As capsaicin desensitization plays a role in pain relief, the present results can help to inform research regarding the therapeutic potential of capsaicin. These findings also highlight the heterogeneity that exists across the oral cavity and have important relevance for understanding the flavor experience of eating spicy foods.

Sensitivity differences to capsaicin across two cultural groups were also explored in the present study. Between American Caucasians and Indians (and males and females), there does not appear to be a difference in capsaicin sensitivity after chili pepper affinity factors were controlled for. These findings provide insights for subject recruitment during sensory tests involving spicy flavors and foods. While chili pepper affinity may still impact perceived spiciness and therefore need to be addressed during recruitment selection, demographics such as ethnicity and sex may not be as high of importance. The subject-matching design utilized here also has utility for future sensory testing aimed at

63 investigating the impact of various subject characteristics on metrics including sensitivity, liking, or preference.

References

National Center for Biotechnology Information. PubChem Compound Database; CID=1548943, (accessed Mar. 7, 2019).

Barbero G, Molinillo J. M. Varela R, et al. (2010). Application of Hansch’s Model to Capsaicinoids and Capsinoids: A Study Using the Quantitative Structure-Activity Relationship. A Novel Method for the Synthesis of Capsinoids, 58(6):3342-9.

Barral, J.P., Croiber, A. (2007). Manual Therapy for the Peripheral Nerves (1st ed). Churchill Livingstone Elsevier.

Bartoshuk, L. M., Duffy, V. B., Green, B. G., Hoffman, H. J., Ko, C.-W., Lucchina, L. A., Weiffenbach, J. M. (2004). Valid across-group comparisons with labeled scales: The gLMS versus magnitude matching. Physiology & Behavior, 82(1), 109–114. (Caterina and Schumacher 1997)

Berger, A., Henderson, M., Nadoolman, W., Duffy, V., Cooper, D., Saberski, L, . . . Bartoshuk, L. (1995). Oral capsaicin provides temporary relief for oral mucositis pain secondary to chemotherapy/radiation therapy. Journal of Pain and Symptom Management, 10(3):243–248. https://doi.org/10.1016/0885-3924(94)00130-D

Bertino, M., & Chan, M. M. (1986). Taste perception and diet in individuals with Chinese and European ethnic backgrounds. Chemical Senses, 11(2), 229–241. https://doi.org/10.1093/chemse/11.2.229

Carr, M.J., Kollarik, M., Meeker, S.N., Undem, B.J. (2003). A Role for TRPV1 in Bradykinin-Induced Excitation of Vagal Airway Afferent Nerve Terminals. Journal of Pharmacology and Experimental Therapeutics 304(3):1275–1279. https://doi.org/10.1124/jpet.102.043422

Carstens, E., Albin, K. C., Simons, C. T., & Carstens, M. I. (2007). Time Course of Self- Desensitization of Oral Irritation by Nicotine and Capsaicin. Chemical Senses, 32(9), 811–816. https://doi.org/10.1093/chemse/bjm048

Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature, 389(6653):816-824. https://doi.org/10.1038/39807

Cliff, M. A., & Green, B. G. (1996). Sensitization and desensitization to capsaicin and menthol in the oral cavity: Interactions and individual differences. Physiology & Behavior, 59(3), 487–494. https://doi.org/10.1016/0031-9384(95)02089-6

Cohen, B.A. (2013). Oral Cavity. In: Cohen BA (ed). Pediatric. Saunders Elsevier. (p. 240-263).

Dessirier, J.M. (2000). Sensory Properties of Citric Acid: Psychophysical Evidence for Sensitization, Self-desensitization, Cross-desensitization and Cross-stimulus- induced Recovery Following Capsaicin. Chemical Senses, 25(6):769–780. https://doi.org/10.1093/chemse/25.6.769

Dessirier, J.M., O’Mahony, M., & Carstens, E. (1997). Oral Irritant Effects of Nicotine: Psychophysical Evidence for Decreased Sensation Following Repeated Application and Lack of Cross-desensitization to Capsaicin. Chemical Senses, 22(5):483–492. https://doi.org/10.1093/chemse/22.5.483

Dessirier, J.M., Simons, C.T., Sudo, M., Sudo S., & Carstens, E. (2000). Sensitization, Desensitization and Stimulus-Induced Recovery of Trigeminal Neuronal Responses to Oral Capsaicin and Nicotine. Journal of Neurophysiology, 84(4):1851–1862. https://doi.org/10.1152/jn.2000.84.4.1851

Fattori, V., Hohmann, M., Rossaneis, A., Pinho-Ribeiro, F.A., Verri, W.A. (2016). Capsaicin: Current Understanding of Its Mechanisms and Therapy of Pain and Other Pre-Clinical and Clinical Uses. Molecules, 21(7):844. https://doi.org/10.3390/molecules21070844

Gazerani, P., Andersen, O. K., & Arendt-Nielsen, L. (2005). A human experimental capsaicin model for trigeminal sensitization. Gender-specific differences: Pain, 118(1), 155–163. https://doi.org/10.1016/j.pain.2005.08.009

Gazerani, P., & Arendt-Nielsen, L. (2005). The impact of ethnic differences in response to capsaicin-induced trigeminal sensitization: Pain, 117(1), 223–229. https://doi.org/10.1016/j.pain.2005.06.010

González-Ramírez, R., Chen, Y., Liedtke, W.B., Morales-Lázaro, S.L. (2017). TRP Channels and Pain. In: Emir, T.L.R. (Ed.). Neurobiology of TRP Channels. Boca Raton: CRC Press/Taylor & Francis.

Green, B.G. (1998). Capsaicin Desensitization and Stimulus-Induced Recovery on Facial Compared to Lingual Skin. Physiology & Behavior, 65(3):517–523. https://doi.org/10.1016/S0031-9384(98)00202-9

Green, B.G. (1996). Rapid recovery from capsaicin desensitization during recurrent stimulation: Pain, 68(2):245–253. https://doi.org/10.1016/S0304-3959(96)03211- 3

Green, B.G. (1991). Temporal characteristics of capsaicin sensitization and desensitization on the tongue. Physiology & Behavior, 49(3):501–505. https://doi.org/10.1016/0031-9384(91)90271-O

Green, B.G. (1989). Capsaicin sensitization and desensitization on the tongue produced by brief exposures to a low concentration. Neuroscience Letters 107(1-3):173– 178. https://doi.org/10.1016/0304-3940(89)90812-4

Green, B. G. (1986). Sensory interactions between capsaicin and temperature in the oral cavity. Chemical Senses, 11(3), 371–382. https://doi.org/10.1093/chemse/11.3.371

Green, B. G., Shaffer, G. S., & Gilmore, M. M. (1993). Derivation and evaluation of a semantic scale of oral sensation magnitude with apparent ratio properties. Chemical Senses, 18(6), 683–702. https://doi.org/10.1093/chemse/18.6.683

Hayes, J.E. (2000). Psychophysical and physiological response of humans to the oral irritant capsaicin. MS thesis. Cornell University.

Hernandez, S.V., & Lawless, H.T. (1998). A method of adjustment for preferred levels of capsaicin in liquid and solid food systems among panelists of two ethnic groups and comparison to hedonic scaling. Food Quality and Preference 10(1):41–49. https://doi.org/10.1016/S0950-3293(98)00036-6

Higgins, M.J., Hayes, J.E. (2019). Regional Variation of Bitter Taste and Aftertaste in Humans. Chemical Senses, 44(9):721–732. https://doi.org/10.1093/chemse/bjz064

Huff, T., Daly, D.T. Neuroanatomy, Cranial Nerve 5 (Trigeminal) [Updated 2019 Apr 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-.

Jessell, T.M., Iversen, L.L., & Cuello, A.C. (1978). Capsaicin-induced depletion of substance P from primary sensory neurons. Brain Research, 152(1):183-188. https://doi.org/10.1016/0006-8993(78)90146-4

Karrer, T., & Bartoshuk, L. (1991). Capsaicin desensitization and recovery on the human tongue. Physiology & Behavior, 49(4), 757–764. https://doi.org/10.1016/0031- 9384(91)90315-F

Kido, M.A., Muroya, H., Yamaza, T., Terada, Y., Tanaka, T. (2003). Vanilloid Receptor Expression in the Rat Tongue and Palate. Journal of Dental Research, 82(5):393– 397. https://doi.org/10.1177/154405910308200513

Kawashima, M., Imura, K., Sato, I. (2012). CGRP-immunoreactive nerve terminals in rodent tongue. European Journal of Histology. 56(2):e21. https://doi.org/ 10.4081/ejh.2012.21 67

Kenins, P. (1982). Responses of single nerve fibres to capsaicin applied to the skin. Neuroscience Letters, 29(1):83–88. https://doi.org/10.1016/0304-3940(82)90369- X

Koplas, P.A., Rosenberg, R.L., Oxford, G.S. (1997). The Role of Calcium in the Desensitization of Capsaicin Responses in Rat Dorsal Root Ganglion Neurons. The Journal of Neuroscience, 17(10):3525–3537. https://doi.org/10.1523/JNEUROSCI.17-10-03525.1997

Kosuge, S., Furuta, M. (1970). Studies on the Pungent Principle of Capsicum: Part XIV Chemical Constitution of the Pungent Principle †. Agricultural and Biological Chemistry, 34(2):248–256. https://doi.org/10.1080/00021369.1970.10859594

Kalantzis, A., Robinson, P.P., Loescher, A.R. (2007). Effects of capsaicin and menthol on oral thermal sensory thresholds. Archives of Oral Biology, 52(2):149–153. https://doi.org/10.1016/j.archoralbio.2006.09.001

Katrinzky, A.R., Xu Y.J., Vakulenko A.V., Wilcox A.L., Bley K.R. (2003). Model compounds of caged capsaicin: design synthesis, and photoreactivity. Journal of Organic Chemistry, 68(23):9100-9104. https://doi.org/10.1021/jo034616t

Lawless, H.T., Rozin, P., & Shenker, J. (1985). Effects of oral capsaicin on gustatory, olfactory and irritant sensations and flavor identification in humans who regularly or rarely consume chili pepper. Chemical Senses, 10(4), 579–589. https://doi.org/10.1093/chemse/10.4.579

Lawless, H.T., Stevens, D.A. (1988). Responses by humans to oral chemical irritants as a function of locus of stimulation. Perception & Psychophysics 43(1):72–78. https://doi.org/10.3758/BF03208975

Lee, S.K., Lee, S.W., Chung, S.C., Kim, Y.K., & Kho, H.S. (2002). Analysis of residual saliva and minor salivary gland secretions in patients with dry mouth. Archives or Oral Biology, 47(9):637–641. https://doi.org/10.1016/s0003-9969(02)00053-5

Lesch, C.A., Squier, C.A., Cruchley, A., Williams, D.M., & Speight, P. (1989). The Permeability of Human Oral Mucosa and Skin to Water. Journal of Dental Research, 68(9):1345–1349. https://doi.org/10.1177/00220345890680091101

Lishko, P.V., Procko, E., Jin, X., Phelps, C.B., Gaudet, R. (2007). The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron. 54(6):905-918.

Liu, L., Simon, S.A. (1996). Capsaicin-induced currents with distinct desensitization and Ca2+ dependence in rat trigeminal ganglion cells. Journal of Neurophysiology 75(4):1503–1514. https://doi.org/10.1152/jn.1996.75.4.1503

López-Carnllo, L., Avila, M. H., & Dubrow, R. (1994). Chili Pepper Consumption and Gastric Cancer in Mexico: A Case-Control Study. American Journal of Epidemiology, 139(3), 263–271. https://doi.org/10.1093/oxfordjournals.aje.a116993

Ludy, M.-J., & Mattes, R. D. (2012). Comparison of sensory, physiological, personality, and cultural attributes in regular spicy food users and non-users. Appetite, 58(1), 19–27. https://doi.org/10.1016/j.appet.2011.09.018

Ludy, M.-J., & Mattes, R. D. (2011). The effects of hedonically acceptable red pepper doses on thermogenesis and appetite. Physiology & Behavior, 102(3–4), 251–258. https://doi.org/10.1016/j.physbeh.2010.11.018

Marsh, S.J., Stansfield, D.A., Brown, D.A., Davey, R., McCarthy, D. (1987). The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro. Neuroscience, 23(1):275-289.

Mcburney, D.H., Balaban, C.D., Christopher, D.E., Harvey, C. (1997). Adaptation to Capsaicin Within and Across Days. Physiology & Behavior 61(2):181–190. https://doi.org/10.1016/S0031-9384(96)00366-6

Nolden, A.A., Hayes, J.E. (2017). Perceptual and affective responses to sampled capsaicin differ by reported intake. Food Quality and Preference, 55:26–34. https://doi.org/10.1016/j.foodqual.2016.08.003

Prescott, J. (1999). The Generalizability of Capsaicin Sensitization and Desensitization. Physiology & Behavior, 66(5):741–749. https://doi.org/10.1016/S0031- 9384(99)00012-8.

Prescott, J., Swain-Campbell, N. (2000). Responses to Repeated Oral Irritation by Capsaicin, Cinnamaldehyde and Ethanol in PROP Tasters and Non-tasters. Chemical Senses 25(3):239–246. https://doi.org/10.1093/chemse/25.3.239

Rentmeister-Bryant, H., Green, B.G. (1997). Perceived Irritation during Ingestion of Capsaicin or Piperine: Comparison of Trigeminal and Non-trigeminal Areas. Chemical Senses, 22(3):257–266. https://doi.org/10.1093/chemse/22.3.257

Reyes-Escogido, M., Gonzalez-Mondragon, E.G., Vazquez-Tzompantzi, E. (2011). Chemical and Pharmacological Aspects of Capsaicin. Molecules, 16(2):1253– 1270. https://doi.org/10.3390/molecules16021253

Rosenbaum, T., Simon, SA. (2007). TRPV1 Receptors and Signal Transduction. In: Liedtke WB, Heller S, editors. TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. Boca Raton: CRC Press/Taylor & Francis.

Rozin, P., Schiller, D. (1980). The nature and acquisition of a preference for chili pepper by humans. Motivation and Emotion, 4(1):77–101. https://doi.org/10.1007/BF00995932

Sanz-Salvador, L., Andrés-Borderia, A., Ferrer-Montiel, A., & Planells-Cases, R. (2012). Agonist- and Ca 2+ -dependent Desensitization of TRPV1 Channel Targets the Receptor to Lysosomes for Degradation. Journal of Biological Chemistry, 287(23), 19462–19471. https://doi.org/10.1074/jbc.M111.289751

Schneider, D., Seuß-Baum, I., Schlich, E. (2015). Determination of Thresholds for Capsaicin in Aqueous and Oil-Based Solutions. In: Guthrie B, Beauchamp J, Buettner A, Lavine BK (Eds.), The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration. American Chemical Society, (pp 171–182), Washington, DC.

Silvestre, F.J., Silvestre-Rangil, J., Tamarit-Santafe, C., & Bautista, D. (2012). Application of a capsaicin rinse in the treatment of burning mouth syndrome. Medicina Oral Patología Oral y Cirugia Bucal, 17(1):e1–e4. https://doi.org/10.4317/medoral.17219

Siruguri, V., & Bhat, R. V. (2015). Assessing intake of spices by pattern of spice use, frequency of consumption and portion size of spices consumed from routinely prepared dishes in southern India. Nutrition Journal, 14(1). https://doi.org/10.1186/1475-2891-14-7

Stevens, D., Lawless, H. (1987). Enhancement of responses to sequential presentation of oral chemical irritants. Physiology and Behavior. 39(1):63–65. https://doi.org/10.1016/0031-9384(87)90344-1

Stevenson, R.J., Prescott, J. (1994). The effects of prior experience with capsaicin on ratings of. Chemical Senses, 19(6):651-656. https://doi.org/10.1093/chemse/19.6.651

Stevenson, R. J., & Yeomans, M. R. (1993). Differences in ratings of intensity and pleasantness for the capsaicin burn between chili likers and non-Iikers; implications for liking development. Chemical Senses. 18(5): 471-482.

Svensson, P., Bjerring, P., Arendt-Nielsen, L, & Kaaber, S. (1993). Sensory and pain thresholds to orofacial argon laser stimulation in patients with chronic burning mouth syndrome. Clinical Journal of Pain, 9(3):207-215. https://doi.org/ 10.1097/00002508-199309000-00009

Szallasi, A., Blumberg, P.M. (1992). Vanilloid receptor loss in rat sensory ganglia associated with long term desensitization to resiniferatoxin. Neuroscience Letters, 140(1):51–54. https://doi.org/10.1016/0304-3940(92)90679-2

Szolcsányi, J. (1977). A pharmological approach to elucidation of the role of different nerve fibres and receptor endings in mediation of pain. Journal of Physiolology (Paris), 73(3):251-259.

Thomas, B.V., Schreiber, A.A., Weisskopf, C.P. (1998). Simple Method for Quantitation of Capsaicinoids in Peppers Using Capillary Gas Chromatography. Journal of Agricultural and Food Chemistry, 46(7):2655–2663. https://doi.org/10.1021/jf970695w

Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen T.A., Gilbert H., Skinner K, . . .Julius D. (1998). The Cloned Capsaicin Receptor Integrates Multiple Pain- Producing Stimuli. Neuron, 21:531–543. https://doi.org/10.1016/S0896- 6273(00)80564-4

Trevisani, M., Smart, D., Gunthorpe, M.J., Tognetto, M., Barbieri, M., Campi, B, . . . Geppetti, P. (2002). Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1. Nature Neuroscience, 5(6):546-551. https://doi.org/10.1038/nn0602-852

Vera-Guzmán AM, Aquino-Bolaños EN, Heredia-García E, Carrillo-Rodriguez J., Hernandez-Delgado S., Chavez-Servia, J.L. (2017). Flavonoid and Capsaicinoid Contents and Consumption of Mexican Chili Pepper (Capsicum annuum L.) Landraces. In: Justino G.C. (Ed.) Flavonoids - From Biosynthesis to Human Health. InTech.

Virus, R.M., Gebhart, G.F. (1979). Pharmacologic Actions of Capsaicin: Apparent Involvement of Substance P and Serotonin. Life Sciences, 25(15):1273-1284. https://doi.org/10.1016/0024-3205(79)90392-8

Wertz, P.W., Swartzendruber, D.C., Squier, C.A. (1993). Regional variation in the structure and permeability of oral mucosa and skin. Advanced Drug Delivery Reviews, 12(1-2):1–12.

Winning, T.A., Townsend, G.C. (2000). Oral mucosal embryology and histology. Clinics in Dermatology, 18(5):499–511. https://doi.org/10.1016/S0738-081X(00)00140-1

Zhang, X., Daugherty, S.L., de Groat, W.C. (2011). Activation of CaMKII and ERK1/2 contributes to the time-dependent potentiation of Ca 2+ response elicited by repeated application of capsaicin in rat DRG neurons. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 300(3):R644– R654. https://doi.org/10.1152/ajpregu.00672.2010

Zygmunt, P.M., Petersson, J., Andersson, D.A., Chuang, H, Sørgård, M., Di Marzo, V., Julius, D., Högestätt ED. (1999). Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature, 400(6743):452–457. https://doi.org/10.1038/22761

Appendix A: Consent Form

Appendix B: Subject Questionnaire