The Effect of Menthol on Nicotine Metabolism

Home , Menthol

THE EFFECT OF MENTHOL ON NICOTINE METABOLISM:

A CROSS SPECIES EVALUATION

Wendy Lee Pace

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

December 2013

APPROVED:

Duane B. Huggett, Major Professor Mark Burleson, Committee Member Teresa D. Golden, Committee Member Sam Atkinson, Interim Chair of the Department of Biological Sciences Mark Wardell, Dean of the Toulouse Graduate School Pace, Wendy Lee. The effect of menthol on nicotine metabolism: A cross

species evaluation. Master of Science (Biology) December 2013, 75 pp., 3 tables, 15

figures, references, 208 titles.

The effect of menthol on nicotine metabolism was examined in liver S9 fractions

of four different species and in the in vivo mouse model. The purpose of this study was

to investigate three parameters: (1) biotransformation of nicotine to cotinine in various

species (human, mouse, rat and trout) using in vitro methods; (2) to determine if the

addition of menthol with nicotine altered biotransformation of nicotine to cotinine; (3) and

to assess similar parameters in an in vivo mouse model. The major findings of this

study include: (1) mice appear to metabolize nicotine, over time, in a manner similar to

humans; (2) menthol decreased cotinine production, over time, after a single dose in mice; and (3) menthol increased cotinine production, over time, after repeated doses, in mice.

Wendy Lee Pace

ii ACKNOWLEDGEMENTS

I gratefully acknowledge the opportunity and financial and academic support of

Dr. Duane Huggett, without whom, this research would not have been possible. Special thanks to Dr. David Hala and Dr. Lene Petersen for everything you both have done.

Thank you, also, to my committee members, Dr. Mark Burleson and Dr. Teresa Golden, for your participation in this process. It is greatly appreciated!

I would also like to thank my parents, Dale and Dawn Orr, and grandparents,

Homer and JoAnn Day.

Funding for this project was provided by Lorillard, Inc., Greensboro, NC.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... i

LIST OF TABLES ...... iv

LIST OF FIGURES ...... v

CHAPTER 1. INTRODUCTION ...... 1 1.1 General Background ...... 1 1.2 The Cytochrome P450 (CYP) System ...... 2 1.3 Nicotine ...... 3 1.3.1 Absorption, Distribution, Metabolism and Elimination (ADME) ...... 3 1.3.2 Chemical Properites ...... 5 1.4 Cotinine ...... 6 1.4.1 ADME ...... 6 1.4.2 Chemical Properties ...... 7 1.5 Menthol ...... 7 1.5.1 ADME ...... 8 1.5.2 Chemical Properties ...... 9 1.6 Model Organisms ...... 10 1.7 Rationale for Study ...... 11 1.8 Objectives and Hypotheses ...... 12

CHAPTER 2. GC/MS ANALYSIS OF TARGET ANALYTES ...... 14 2.1 Introduction ...... 14 2.2 Methods ...... 16 2.2.1 GC/MS ...... 16 2.2.2 Extraction Methods ...... 19 2.3 Results ...... 20 2.3.1 Standard Curve ...... 20 2.3.2 Enzyme Activity ...... 23 2.3.3 Extraction Solvent Comparison ...... 24 2.4 Discussion ...... 25

ii CHAPTER 3. NICOTINE METABOLISM IN S9 FRACTIONS ...... 28 3.1 Introduction ...... 28 3.2 Materials and Methods...... 32 3.2.1 Chemicals ...... 32 3.2.2 S9 Fractions ...... 32 3.2.3 Assay and Sample Preparation ...... 33 3.3 Results ...... 34 3.3.1 Human, Rat and Trout ...... 34 3.3.2 Human S9 Assay ...... 35 3.3.3 Mouse S9 Assay ...... 36 3.3.4 Statistical Analysis ...... 39 3.4 Discussion ...... 39

CHAPTER 4. NICOTINE METABOLISM IN THE MOUSE MODEL ...... 44 4.1 Introduction ...... 44 4.2 Materials and Methods...... 47 4.2.1 Model Animals...... 47 4.2.2 Chemicals ...... 47 4.2.3 Acute Single-Dose ...... 48 4.2.4 Repeated Dose 14-day ...... 48 4.2.5 Sample Prep ...... 49 4.3 Results ...... 49 4.4 Statistical Analysis ...... 51 4.5 Discussion ...... 51

CHAPTER 5 CONCLUSIONS AND SUMMARY ...... 58

REFERENCES ...... 60

iii LIST OF TABLES

Page

Table 1.1 Physical and Chemical Properties of Nicotine, Cotinine and Menthol ...... 10

Table 2.1 Selected Ions and Retention Times *Indicates retention time observed with Agilent 6890N vs. 5890 HP Series II ...... 16

Table 3.1 CYP2A6 Substrates *Table adapted from Raunio et al., 2001. (Chang and Waxman, 1996; Pelkonen et al., 2000; Raunio et al., 1997; Mandan et al., 1995; Sadeque et al., 1997; Hong et al., 1999; Miyazawa et al., 2011) ...... 29

iv LIST OF FIGURES

Page

Figure 1.1. Primary routes of nicotine metabolism. Image adapted from Hukkanen et al. (2005)...... 4

Figure 1.2. Proposed menthol metabolic pathway. Image adapted from Speijers (1999)...... 8

Figure 2.1. Basic block diagram of typical GC/MS. Image adapted from FAO/WHO (2006)...... 15

Figure 2.2. Ion spectra for nicotine. Image shows quantifying ion (162) and qualifying ion (133). Spectra captured from instrument operated in SIM mode...... 17

Figure 2.3. Ion spectra for cotinine. Image shows quantifying ion (176) and qualifying ion (118). Spectra captured from instrument operated in SIM mode...... 18

Figure 2.4. Nicotine standard curve. Graph shows standard curve for nicotine with r² = 0.985...... 21

Figure 2.5. Cotinine standard curve. Graph shows standard curve for cotinine with r² = 0.982...... 22

Figure 2.6. Nicotine and cotinine chromatogram. Chromatograms show peaks and retention times for nicotine (9.50) and cotinine (12.92)...... 23

Figure 2.7. Metabolic activity assay. Graph shows nicotine and cotinine concentrations from a metabolic assay with human S9 fractions (Moltox). Data shows no decrease in nicotine production and no increase in cotinine production (± standard deviation)...... 24

Figure 2.8. Extraction solvent comparison assay. Graph represents differences in analyte recovery between two solvents, acetone and acetonitrile (ACN) Solvent dependent recovery shows greater variability in cotinine recovery with acetone as compared to acetonitrile (ACN). Nicotine recovery is greatly reduced with acetone and ACN as compared to the check standard (± standard deviation)...... 25

Figure 3.1. Human, rat and trout S9 assay. Loss of nicotine following the administration of nicotine at 10 ppm in three species of liver S9 fractions, human (Moltox) trout (in house preparation) and rat (Moltox) (± standard deviation). *Indicates significant (p ≤ 0.05) difference between species...... 35

Figure 3.2. Human S9 assay. Data shows formation of cotinine in pooled human liver S9 fractions (Xenotech) following the administration of nicotine at 10 ppm or nicotine 10 ppm + menthol 1 ppm (± standard deviation). *Indicates significant (p ≤ 0.05) difference between control (nicotine 10 ppm) and menthol co-exposure. Dissimilar letters indicate significant (p ≤ 0.05) difference between first time point and other time points within each

v group (significant change from (a) to (b) and also, from (b) to (c)). +Indicates time point without a replicate value...... 36

Figure 3.3. Mouse S9 assay. Formation of cotinine in pooled mouse liver S9 fractions (Moltox) following administration of nicotine only (10 ppm) or co-administration with varying concentrations of menthol; A) 1 ppm, B) 0.5 ppm, C) 0.25 ppm, D) 0.1 ppm, E) 0.01 ppm (± standard deviation). *Indicates significant (p ≤ 0.05) difference between control (nicotine only) and menthol co-exposures. Dissimilar letters indicate significant (p ≤ 0.05) differences between first time point and other time points within each group (significant change from (a) to (b) and also, from (b) to (c))...... 39

Figure 4.1. Acute single dose. Data shows average plasma concentrations of cotinine (ppm) in mice following a single dose regime with nicotine 1 mg/kg, nicotine 1 mg/kg + menthol 0.5 mg/kg or nicotine + menthol 0.01 mg/kg (± standard deviation). *Indicates significant (p ≤ 0.05) difference between acute and 14 day dosing schedule...... 50

Figure 4.2. 14-Day repeated dose. Data shows average plasma concentrations of cotinine (ppm) in mice following a 14 day (1 dose per day) dose regime with nicotine 1 mg/kg, nicotine 1 mg/kg + menthol 0.5 mg/kg or nicotine + menthol 0.01 mg/kg (± standard deviation). *Indicates significant (p ≤ 0.05) difference between acute and 14- day dosing schedule...... 51

vi CHAPTER 1

INTRODUCTION

1.1 General Background

Cigarette use is commonly linked with a broad range of adverse health effects including increased incidence of cancers, heart disease, diabetes, changes in thyroid and cortisol levels, and an overall acceleration of the aging process (Newcome and

Carbone, 1992; Benowitz, 2003; Ezzati et al., 2005; Kapoor and Jones, 2005; Bernhard et al., 2007). Recently, it was noticed that the demographic with the highest incidence of disease also tended to smoke mentholated cigarettes (Wagenknecht et al., 1990;

Gardiner, 2004; Ahijevych et al., 1996; Perez-Stable et al., 1998). This observation tentatively suggests a correlation between this type of cigarette and health effects. It has further been reported that a higher percentage of African American smokers prefer mentholated cigarettes as opposed to Caucasian smokers who tend to prefer non- mentholated cigarettes (Cummings et al., 1987; Sydney et al., 1989). It was theorized that the addition of menthol into cigarettes may enhance toxicity of cigarette smoke leading to increased incidence of cancer in these individuals (Cummings et al., 1987;

Novotny et al., 1988; Sidney et al., 1989 and 1995). Although a direct increase of cancer risk from the addition of menthol has not been documented and is now ruled out, information regarding this relation is still lacking (Heck, 2010). Currently, only a few studies have looked at the effect of mentholated cigarettes in pathologies that are not cancer related (Clark et al., 2004).

1 1.2 The Cytochrome P450 (CYP) System

This system is responsible for the bulk of xenobiotic detoxification and bioactivation. It is a catalytic cycle of monooxygenase reactions leading to the eventual

processing and elimination of various compounds. The greatest concentration of these

CYP proteins is located in the liver endoplasmic reticulum of humans and many other

species. P450s are named based on the red pigment produced by their heme co-factor

group. When in the ferrous state (reduced, Fe²⁺) and bound to CO, the complex will

absorb light maximally at 450 nm. Although this is an inactive state, it is readily

reversed (Klaassen, 2001).

Many advances have been made in the past 20 years leading to the discovery of

57 genes (58 pseudogenes) in the human system. Approximately a quarter of these remain “orphan” genes due to inadequate information about their function. These genes have been placed into families and are generally thought of in terms of the substrates they utilize. Those grouped with the prefix of either 1, 2 or 3 being the proteins responsible for most xenobiotic transformation (Guengerich, 2008).

A valuable consequence of identifying the function of these individual proteins is

the ability to predict toxicity and/or bioavailability of a drug. A well known example of toxicity is furanocoumarins in grapefruit juice inhibiting CYP3A4 leading to increased plasma levels of certain drugs with possible adverse effects, including patient death

(Guo et al., 2004). An example of bioactivation is the commonly prescribed and

abused, carisoprodol. Carisoprodol is a pro-drug that must be activated by CYP2C19 to

become the biologically active compound meprobamate that was regularly prescribed in

the late 50s (Bramness et al., 2003).

2 1.3 Nicotine

Humans have used nicotine as a compound of recreational use for centuries.

Nicotine is primarily inhaled in the form of tobacco smoking. The compound is a natural

alkaloid occurring at about 1.5% in tobacco leaves with approximately 1-1.5 mg

absorbed during the course of smoking (Kozlowski et al., 1998; Benowitz and Jacob,

1984). Although the number of smokers has declined from 1997 to 2009, there are still

approximately 443,000 deaths and $193 billion in lost productivity and direct health-care

costs each year (CDC, 2011; CDC, 2008). The African American demographic repeatedly shows a higher incidence of disability, cancer, and death attributed to cigarette smoking than Caucasian smokers (CDC, 2011; CDC, 2008). It has been

shown that the African American preference for mentholated cigarettes versus non-

mentholated is 70% compared to Caucasians at 30% for each group of smokers

(USDHHS, 1998). Cotinine, the major metabolite of nicotine, remains at higher levels in

African American smokers in spite of their lower level of cigarette consumption per day

and decreased puff rate as compared with their Caucasian counterparts (Wagenknecht

et al., 1990; Gardiner, 2004; Benowitz et al., 1999; Carabello et al., 1998; Clark et al.,

1996; McCarthy et al., 1996). Mechanisms influencing the differences in metabolism of

cigarette smoke and the development of disease in these two demographics have not

been established.

1.3.1 Absorption, Distribution, Metabolism and Elimination (ADME)

It is estimated that approximately 80-90% of inhaled nicotine is absorbed in the

alveoli and small airways (Benowitz and Jacob, 1984; Armitage et al., 1975; Gori and

3 Lynch, 1985). Incoming smoke, at a pH of 5.5-6.0 (or higher in European cigarettes), carries the majority of ionized nicotine past the buccal cavity into the lung fluid (pH 7.4), to reach the brain in 10-20 seconds (Sensabaugh and Cundiff, 1967; Brunnemann and

Hoffmann, 1974; Gori et al., 1986; Benowitz et al., 1990; Benowitz, 1996b). According to various autopsy results on smokers, nicotine is distributed extensively throughout the body with the highest affinities and accumulations appearing to be within tissues of the liver, kidney, spleen, and lungs (Urakawa et al., 1994).

Figure 1.1. Primary routes of nicotine metabolism. Image adapted from Hukkanen et al. (2005).

Nicotine metabolism is well studied and appears to be dependent on CYP2A6

gene control of P450 enzymes (Nakajima et al., 1996a; Nakajima et al., 1996b;

Nakajima et al., 2000). After absorption, nicotine is transformed into various

metabolites primarily within the liver. The transformation involves two main steps. The

4 first step results in nicotine-∆1’(5’)-iminium ions in equilibrium with 5’-hydroxynicotine catalyzed by cytochrome P450 (Murphy, 1973; Brandage and Lindblom, 1976b;

Peterson et al., 1987). The second step is catalization by a cytoplasmic aldehyde oxidase (Brandage and Lindblom, 1979a; Gorrod and Hibberd, 1982). Cotinine is the

primary metabolite produced in humans and most mammals, including mice, with

approximately 70-80% of nicotine converted to cotinine in humans (Benowitz and

Jacob, 1994). Due to the extended half-life of cotinine compared with nicotine, cotinine

is commonly used as the biomarker for nicotine metabolism (Benowitz, 1996a;

Hukkanen et al., 2005).

1.3.2 Chemical Properites

Nicotine [1-methyl-2-(3-pyridyl-pyrrolidine), C10H14N2] is an alkaloid product

produced by members of the solanoceous plant family. The tobacco plant (Nicotiana

tabacum) is part of this family (Pictet and Crepieux, 1895; Doolittle et al., 1995).

Nicotine is a yellow oily liquid with a molecular weight of 162.12 with melting/boiling

points at -79ºC and 247ºC, respectively. The structure is comprised of pyridine and

pyrrolidine rings and has the ability to cross the blood brain barrier (Schevelbein, 1982;

Yildez et al., 1988). Nicotine circulates largely unbound to plasma proteins (>5%) with

approximately 69% of the product ionized and 31% unionized (Benowitz et al., 1982).

Tobacco contains predominately the (S)-isomer form, although, the (R)-isomer is slightly

higher (up to 10%) in tobacco smoke (Armstrong, 1998; Klus and Kuhn, 1997; Pool et

al., 1985). In humans and mice, nicotine is metabolized by CYP2A6 and CYP2A5

5 respectively, with cotinine being the major metabolite (Nakajima et al., 1996a; Nakajima et al., 1996b; Berkman et al., 1995).

1.4 Cotinine

Cotinine is also found in the leaves of tobacco plants but mainly functions as the major metabolite of nicotine biotransformation in humans. It is used as the biomarker for nicotine exposure during cigarette smoking due to its extended half-life as compared to nicotine (Benowitz, 1996a). Cotinine has been shown to be biologically active stimulating acetylcholine nicotinic receptors, although, this action is minimal when compared with nicotine (Briggs and McKenna, 1998; Buccafusco et al., 2007; Dwoskin et al., 1999).

1.4.1 ADME

Cotinine is produced mainly as a metabolite of nicotine ingestion or inhalation at about 75% of the nicotine dose. Six metabolites of cotinine have been identified: 3’- hydroxycotinine, 5’-hydroxycotinine, cotinine N-oxide, cotinine methonium ion, cotinine glucuronide and norcotinine with 3’-hydroxycotinine and its glucuronide conjugate as the most common metabolite found (40-60%) in the urine of smokers (Bowman and

McKennis,1962; McKennis et al., 1963b; Neurath et al., 1987; Neurath, 1994; Shulgin et al., 1987; Kyerematen et al., 1990b; McKennis et al., 1963a; Curvall et al., 1991;

Caldwell et al., 1992; Bowman et al., 1959; Byrd et al., 1992). Cotinine is excreted unchanged in the urine at 10-15% at a rate of approximately 45 mls per minute with a half-life of about 16 hours (Benowitz et al., 1994).

6 1.4.2 Chemical Properties

Cotinine [(S)-1-methyl-5-(3-pyridyl)-2-pyrrolidinone, C10H12N2O] has a molecular weight of 176.22 with a melting and boiling point at 40-42ºC and 250ºC, respectively.

Cotinine d3 [1-(methyl-d3)-5-(3-pyridinyl)-2-pyrrolidinone, C10D3H9N2O], with a molecular weight of 179.22, was used as the internal standard for sample preparation and GC/MS analysis.

1.5 Menthol

Menthol is obtained naturally from peppermint plants of the Mentha genus and is synthesized commercially to meet the demands for human consumption (OECD SIDS).

Menthol is added to a mix of ingredients to enhance flavors in everything from pharmaceuticals to foods. It was first added to cigarettes in 1925 with the introduction of the Spud brand a year later (Reid, 1993). By the 1970s, mentholated cigarettes had become, and still remain, the preferred cigarette in the African American community

(Gardiner, 2004; USDHHS, 1998; Garten and Falkner, 2001). It has been reported that the mean menthol content across 48 brands of mentholated cigarettes recently obtainable in the U.S. is approaching 4 mg menthol/g tobacco (Celebucki et al., 2005;

Gordon et al., 2011).

Menthol is accepted as a “generally recognized as safe” (GRAS) ingredient for oral and topical applications according to the Flavor and Extract Manufacturers

Association (FEMA). In observing this demographic trend in the African American community, menthol, as a major ingredient, has been questioned as a possible catalyst

7 of increased disease that can be attributed to smoking (Clark et al., 2004; Werley et al.,

2007; Benowitz et al., 2004; Richardson, 1997; Kabat and Hebert, 1991; Kabat and

Hebert, 1994; Fridman et al., 1998).

1.5.1 ADME

Menthol is lipid soluble and when taken orally, it is absorbed from the small intestine and rapidly metabolized (Gelal, 2008; Gelal et al., 1999). Menthol is not affected by pyrolysis of normal cigarette smoking and is carried in mainstream smoke at approximately 30% of the total cigarette content (Jenkins et al., 1970). Of that 30%, approximately 70% is expected to be absorbed at inhalation (Haggard and Greenberg,

1941; Galworski et al., 1997).

Figure 1.2. Proposed menthol metabolic pathway. Image adapted from Speijers (1999).

Menthol, like nicotine, is metabolized by CYP2A6. Alternating reactions of hydroxylization and oxidation followed by glucuronidation produces menthol glucuronide

8 as the major metabolite in humans (Miyazawa et al., 2011; Farco and Grundmann,

2013). The half-life has been shown to be 56.2-46.2 minutes after oral administration and only 11.7 minutes after smoking a mentholated cigarette. Menthol glucuronide recovery has been reported at 65-69% of the total menthol dose and has been found in measurable levels in the plasma and/or urine (Hiki et al., 2011; Gelal et al., 1999;

Ahijevych and Garrett, 2004). Menthol glucuronide is excreted mainly in the urine.

1.5.2 Chemical Properties

Menthol [(1R,2S,5R)-2-isoporopyl-5-methylcyclohexanol, C10H20O] is a

naturally occurring monocyclic terpene alcohol which stimulates TNRP8 cold receptors

producing a cooling effect (Bautista et al., 2007; McKemy, 2007; Schafer et al., 1986).

Menthol has also been shown to exhibit antiseptic, analgesic and antispasmotic effects

in various applications (Grigoleit and Grigoleit, 2005; Farco and Grundemann, 2013).

Menthol inhibits the P450 enzymes, CYP2A6 and UGT2B10, and nicotinic acetylcholine

receptor proteins (Muscat, 2009; Hans et al., 2012; Farco and Grundmann, 2013).

Forming opaque crystals in its pure form, menthol has a molecular weight of 156.27 with

a melting/boiling point at 34º-36ºC and 216ºC, respectively. The dominant natural

isomer, and the form found in most cigarettes, is l-menthol but it can occur as four different pairs of isomers (Eccles, 1994; Chen et al., 2011). L-menthol has been shown to be more pharmacologically active and more effectively metabolized by mammalian systems than d-menthol (Caldwell, 1995). Although menthol is heavily consumed, metabolic mechanisms and effects are only recently being investigated.

9 Table 1.1 Physical and Chemical Properties of Nicotine, Cotinine and Menthol

Property Nicotine Cotinine Menthol CAS# 54-11-5 486-56-6 2216-51-5 Molecular Weight ** 162.23 176.22 156.15 Melting Point + -79 41 79 Boiling Point + 247 250 216 Log P * 0.57 0.075 3.216 Log D * -2.22/0.62 0.01/0.07 3.22/3.22 Water Solubility + 1e+006 mg/L 4.891e+004 mg/L 456 mg/L Henry’s Law 6.831e-9 atm- 1.806e-9 atm- 3.630e-6 atm- Constant + m3/mol m3/mol m3/mol pKa 8.5 Log Kow + 1.17 0.07 3.40 *Predicted ACD/Labs; **CSID, www.chemspider.com; +Predicted EPISuite

1.6 Model Organisms

Rodents are commonly used as model organisms with the data being

extrapolated for human applications. They are easily housed and more accessible than

other mammal models. Various strains are available and can be tailored to the type of

testing required. Rats are preferred to mice in many applications due to their larger size

and ease of handling; however, mice have a major advantage in nicotine metabolic

research with their expression of CYP2A5 (Raunio et al., 2008).

Fisher rats (Rattus norvegicus) are an albino inbred strain regularly used in

nicotine research. Inbred lines typically can provide more effective and consistent data

due to reduced variability between individual animals as compared to outbred strains.

Inbred strains are also desirable in reproducing and confirming findings in subsequent

experiments (Festing, 2010). Rats were chosen for this project to confirm previous

findings of minimal cotinine formation and for comparisons between species.

Swiss Webster mice (Mus musculus) are a reliable general multipurpose model

animal. The CFW strain is an outbred stock created and maintained by Charles River

10 Laboratories from a previously inbred line. They are white albino but carry a black agouti gene. Mice were chosen based on previous studies showing homology of the genes CYP2A6/CYP2A6 between humans and mice (Raunio et al., 2008). It was expected that cotinine production in mice is similar to humans allowing any effect from the addition of menthol to be observed.

Aquatic species, such as rainbow trout (Oncorhynchus mykiss), have become increasingly valuable as model organisms. Rainbow trout have been shown to exhibit lower rates of cancers when compared with rodent models and comparable size studies can be performed at a fraction of the cost (Bailey et al., 1996). The P450 system in trout has been shown to have some similarities to humans with regards to their responses to inducers and inhibitors of metabolic processes (Buhler, 1998; Miranda,

1991). While many CYP enzymes in trout and other aquatic species have yet to be characterized and sequenced, the potential for adequate and less demanding model organisms is great (Buhler, 1998). It is because of the previously mentioned characteristics that trout were chosen as a part of this project. Data showing metabolic similarities or differences between trout and other species can potentially reveal previously unknown mechanisms and/or provide better model organisms for specific tasks.

1.7 Rationale for Study

The interaction of menthol and nicotine and their metabolic effects have not been thoroughly investigated and definitive literature on the subject is quite limited. This lack of information, in spite of the great quantity of mentholated cigarettes consumed, poses

11 many questions about possible health effects of these products. Available literature does report studies of this type based on the incidence of certain types of cancers, however, this is a very narrow view of the possible effects that this may have on other physiological functions (e.g., inflammation, aging, glucose intolerance) (Lerner et al.,

2009; Bernhard et al., 2007; Houston et al., 2006). Cigarette smoking has been

implicated in many disease states (diabetes mellitus, cardiovascular disease, and osteoporosis), not necessarily as a direct cause, but as a preventable complicating factor (Ezzati et al., 2005; Raisz, 2005; Bernhard et al., 2007). In these studies the distinction has not been made between mentholated versus non-mentholated

cigarettes. Studies featuring non-life threatening scenarios can potentially lower health care costs and improve the quality of life for many individuals. Furthermore, due to the lack of a means to consistently and accurately portray human smoking scenarios, attempts to create simpler and more effective methods must be utilized.

To this end, this study investigated three parameters: (1) biotransformation of nicotine to cotinine in various species (human, mouse, rat and trout) using in vitro methods; (2) determined if the addition of menthol with nicotine altered biotransformation of nicotine to cotinine; (3) and assessed similar parameters in an in vivo mouse model.

1.8 Objectives and Hypotheses

Objective 1: Determine if the addition of menthol with nicotine alters the formation of cotinine in mouse liver S9 fractions.

Hypothesis 1: Hₒ: The addition of menthol with nicotine will not significantly alter the formation of cotinine in mouse liver S9 fractions.

12 Objective 2: Determine if the addition of menthol with nicotine alters the formation of cotinine in rat liver S9 fractions.

Hypothesis 2: Hₒ: The addition of menthol with nicotine will not significantly alter the formation of cotinine in rat liver S9 fractions.

Objective 3: Determine if the addition of menthol with nicotine alters the formation of cotinine in trout liver S9 fractions.

Hypothesis 3: Hₒ: The addition of menthol with nicotine will not significantly alter the formation of cotinine in trout liver S9 fractions.

Objective 4: Determine if the addition of menthol with nicotine alters the formation of cotinine in human liver S9 fractions.

Hypothesis 4: Hₒ: The addition of menthol with nicotine will not significantly alter the formation of cotinine in human liver S9 fractions.

Objective 5: Determine if the addition of menthol with nicotine alters the plasma concentration of cotinine in the mouse model over time.

Hypothesis 5: Hₒ: The sub-cutaneous co-administration of menthol with nicotine will not significantly alter the expected plasma levels of cotinine in the mouse model over time.

13 CHAPTER 2

GC/MS ANALYSIS OF TARGET ANALYTES

2.1 Introduction

All experimental samples were analyzed via a gas chromatograph coupled to a quadru pole mass spectrometer (GC/MS). Many options are available; however, due to relative cost and durability, the GC/MS combination is widely used (Vekey, 2001; Dural et al., 2011). This combination of gas chromatography and mass spectrometry offers

good sensitivity in detecting molecules based on volatility.

Gas chromatography separates compounds with the aid of a mobile and

stationary phase. Typically, the mobile phase, a non-reactive carrier gas, will carry the

compounds of interest (analytes) through the stationary phase (column) and allow them

to form separate bands, or zones, based on differences in migration rates of the

analytes. Helium is the carrier gas of choice due to the decreased risk of analyte

ionization while traveling through the instrument. However, recent shortages in supply

have led to the transition to hydrogen or nitrogen.

Columns can be either packed or capillary in structure with the most common for

general use applications being capillary type. The (5% phenyl)-95%methylpolysiloxane composition is non-polar and designed to be non-reactive with basic or acidic compounds making this type of column ideal for general use GC/MS applications. The molecules are bonded, cross-linked and solvent rinsable with very low bleed characteristics. The dominant interaction of polysiloxane lined columns is dispersion.

Separation is achieved based on the differences in melting points of the analytes (e.g. nicotine, -79ºC, and cotinine, 41ºC). Dipole moment and/or hydrogen bonding

14 interactions are weak to non-existent with the addition of phenyl groups and non- existent with the addition of methyl groups (Agilent, 2012).

As analytes pass through and are released from the column, they enter a mass spectrometer (MS). The mass spectrometer ionizes and detects the fragments of the analytes that are produced during the ionization process. Analyte abundance is determined by the retention time and the mass of the fragments. Resulting data can be plotted as a function of time and/or selected-ion monitoring (SIM). The SIM mode limits detection parameters based on mass-to-charge ratios of the analyte, thereby, increasing sensitivity of the instrument (Vekey, 2001). Typical software displays mass chromatograms that allow both formats to be seen concurrently (Skoog, Holler and

Crouch, 2007). The most common method of ionization is electron impact (EI). EI is a hard ionization method resulting in the fragmentation of analytes identified by the detection of positively charged ions.

Figure 2.1. Basic block diagram of typical GC/MS. Image adapted from FAO/WHO (2006).

15 2.2 Methods

2.2.1 GC/MS

Methods for nicotine and cotinine analysis were adapted from Nystrom et al.

(1997) and Davoli et al. (1998). Two different GS/MS instruments were used in the analysis of these data. Mouse data were analyzed on an Agilent 6890N gas chromatograph (Agilent, Palo Alto, CA) coupled with an Agilent 5973 quadrupole mass spectrometer fitted with an EC™-5 capillary column (30 m × 0.25 mm × 0.25 μm)

(Alltech, Deerfield, IL). Human, trout and rat data were analyzed with an Agilent 5890

HP Series II system with a 5972A mass spectrometer fitted with an HP-5MS Agilent

J&W column (30m × 0.25mm × 0.25μm). The temperature range of both columns was

60º-325ºC. The carrier gas used was Ultrapure helium (Air Liquide, Houston, TX). The instruments were operated with an initial flow of 1.2 mL min-1 in constant pressure mode (9.5 psi) with an average velocity of 40 cm sec-1. Auto-injected samples (2.0 μls) were pulsed in splitless mode (Nallini, et al., 2011). Analytes were monitored in selected-ion mode (SIM) and analyzed with the HP ChemStation data program.

Instrument parameters used were as follows: inlet 250ºC, detector 250ºC and oven 80ºC. Ramp conditions were as follows: 15ºC per minute to 250º for 5 minutes,

15ºC per minute to 300ºC for 5 minutes, and then temperature returned to initial conditions. Total run time was 28.62 minutes.

Table 2.1 Selected Ions and Retention Times *Indicates retention time observed with Agilent 6890N vs. 5890 HP Series II

Compound Quantifying Ion Qualifying Ions Retention Time Nicotine 133 84, 162, 161 10.64* - 9.50 Cotinine 176 118, 119, 98 13.89* - 12.92 Cotinine d3 179 101, 122, 176 13.88* - 12.91

Figure 2.2. Ion spectra for nicotine. Image shows quantifying ion (162) and qualifying ion (133). Spectra captured from instrument operated in SIM mode.

Figure 2.3. Ion spectra for cotinine. Image shows quantifying ion (176) and qualifying ion (118). Spectra captured from instrument operated in SIM mode.

18 2.2.2 Extraction Methods

Multiple extraction methods have been reported for the extraction of nicotine and cotinine including liquid/liquid and solid phase extractions (Massadeh et al., 2009;

Davoli et al., 1998). Previous attempts with liquid/liquid and solid phase extraction methods were not successful. Methods were adapted to precipitate particulates out of solution producing a clean sample with minimal loss of analyte.

The human enzyme activity assay (Figure 2.5) was prepared in three separate

volumes that when added together produced a final concentration of 0.5 mg/ml protein,

1 mM NADPH and 10 ppm nicotine. Total volume was adequate to allow two replicate aliquots of 300 µls each to be removed at each time point. Protein volumes were diluted in PBS at pH 7.4. NADPH was prepared with phosphate buffer solution (PBS) at pH 7.4. The nicotine volume was diluted at <1% solvent in DMSO and brought to volume in PBS at pH 7.4. The reaction mix was prepared in glass tubes with the protein

volume added at time 0 to begin the reaction. The assay was run in a water bath

(ThermoScientific 2870) at 37ºC with two 300 µl aliquots removed from each tube at

each time point. Samples were placed into fresh glass tubes with 300 µls cold acetone

to terminate the reaction. Samples were then spiked with cotinine d3 (10 ppm final concentration) as the internal standard and centrifuged for 10-20 minutes at 2000 g to pellet. Supernatant was removed and placed into fresh amber vials. Pellets were reconstituted with 500 µls of cold acetone to rinse pellets and again centrifuged as above with the supernatant removed and placed into corresponding vial. Rinse was performed two times. Samples were allowed to dry to dryness under a gentle stream of nitrogen. Dried samples were reconstituted and rinsed with three replicate 500 µl

19 aliquots of dichloromethane (DCM) and placed into 2 ml amber vials (Thermo Fisher

Scientific, USA). Samples were then concentrated by evaporating to dryness, re-

suspended in DCM and placed in 200 µl glass inserts (Grace Davison Discovery

Sciences, USA) inside 2 ml amber vials for GC/MS analysis.

The extraction solvent assay (Figure 2.6) was prepared in two volumes that when

added together produced a final concentration of 0.5 mg/ml protein (rat) and 10 ppm

nicotine. After both volumes were added together, two replicates of 300 µl aliquots

were immediately removed and placed into fresh glass tubes with either 300 µls cold

acetone or acetonitrile (ACN) to prevent metabolic reaction. Samples were then spiked

with cotinine d3 (10 ppm final concentration) as the internal standard and centrifuged for

10-20 minutes at 2000 g to pellet. Supernatant was removed and filtered through

approximately 2 g sodium sulfate contained in glass Pasteur pipettes and held in place

with glass wool. Filtrate was then concentrated by evaporating to dryness, re-

suspended in DCM and placed in 200 µl glass inserts inside 2 ml amber vials for

GC/MS analysis.

2.3 Results

2.3.1 Standard Curve

An 8 point mixed standard curve was prepared with nicotine, cotinine and

cotinine d3 as the internal standard (Figures 2.2 and 2.3). The calibration curve was

created using serial dilutions from 20 ppm to 0.156 ppm with the internal standard fixed

at 10 ppm. Curve was generated with ChemStation software (Agilent, La Jolla, CA) by

plotting (linear curve fit) the relative response between analyte and internal standard.

20 The two lowest points were excluded creating an optimum quantification range between

20 ppm and 0.625 ppm producing correlation coefficient (r²) values at 0.985 and 0.982 for nicotine and cotinine, respectively. A mixed standard with nicotine (20 ppm), cotinine (20 ppm) and cotinine d3 (10 ppm) was run prior to each set of samples.

Figure 2.4. Nicotine standard curve. Graph shows standard curve for nicotine with r² = 0.985.

Figure 2.5. Cotinine standard curve. Graph shows standard curve for cotinine with r² = 0.982.

Figure 2.6. Nicotine and cotinine chromatogram. Chromatograms show peaks and retention times for nicotine (9.50) and cotinine (12.92).

2.3.2 Enzyme Activity

An assay was performed with human S9 from Moltox to determine P450 enzyme activity and subsequent analyte recovery from a biological matrix. The graph (Figure

2.5) shows no increase in cotinine over time. Nicotine recovery did not decrease over time. The extraction solvent was acetone, leading to approximately 50% recovery of nicotine from the original 10 ppm dose. The lack of decline in nicotine concentrations over time and negligible cotinine recovery suggests that enzymatic activity in this assay was lacking.

23 8 Nicotine 6 Cotinine

Concentration (ppm) 0 30 60 -2 Time (minutes)

2.3.3 Extraction Solvent Comparison

Extraction solvents were compared for optimal analyte recovery. Data (Figure

2.6) shows nicotine and cotinine recovery between the extraction solvents: 1) acetone and (2) acetonitrile (ACN) as compared to a check standard. The check standard was created with a mix of cotinine, nicotine and cotinine d3 (internal standard) at 10 ppm each. Cotinine and nicotine concentrations from the check standard were reported to be approximately 72% and 85% of the prepared concentrations, respectively. Cotinine recovery with acetone and ACN showed greater recovery than the internal standard.

Cotinine recovery showed greater variability than extraction with ACN. Nicotine recovery was higher with the use of acetone than ACN. However, nicotine recoveries were substantially less than the check standard for each solvent, both with high variability.

24 15 Cotinine Nicotine 10

5 Percent Recovery (ppm) 0 CK STD Acetone ACN

2.4 Discussion

Standard curves (Figures 2.2 and 2.3) generated for nicotine and cotinine produced correlation coefficient (r²) values of 0.985 and 0.982, respectively. These values show an adequate linear relationship allowing for sample quantification.

Chromatograms (Figure 2.4) show strong peaks with no tailing and little noise. Limits of

detection (LOD) and limits of quantification (LOQ) values averaging 0.5 ng/ml and 1.42

ng/ml have previously been reported for nicotine and cotinine (Man et al., 2006; Shin et

al., 2006; James et al., 1998; Kim and Huestis, 2006). Instruments used in this study

lacked this level of sensitivity with the LOQ estimated at 0.625 ppm (625 ng/ml);

however, values produced were consistent and within expected parameters. In the

current project, interpretation of results was based on relative values of cotinine

production after nicotine administration compared to relative deviations in cotinine

production with the addition of menthol.

25 Analyte extraction optimization was ongoing throughout the project. Analyte recoveries were less than ideal but still produced consistent and observable trends over time. Nicotine and cotinine recoveries in previous studies have been reported from 86% to well over 100% with extraction methods including: liquid/liquid extraction, solid phase extraction and the now discontinued Toxi-Tubes® (Agilent) (Davoli et al., 1998; Man, et al., 2006; Shin et al., 2002; James et al., 1998; Kim and Huestis, 2006) In the current project, protein precipitation with acetone and nicotine recovery from an S9 assay using human liver purchased from Moltox (Figure 2.5) produced a GC/MS response at approximately 50% of the response produced by the check standard. This substantial loss of analyte most likely occurred during the drying process (Davoli et al., 1998; Man, et al., 2006). Cotinine recovery was negligible. The lack of decrease in nicotine levels coupled with negligible cotinine recovery suggests enzymatic inactivity of the S9 preparation. Subsequent human S9 fractions were purchased from Xenotech and the assay was repeated. Data (Figure 3.2, Chapter 3) shows little change in percent recovery but clearly shows a linear increase in cotinine production indicating enzymatic activity.

Comparison trials between extraction solvents were performed to determine if the use of a different solvent would increase analyte recovery. Initially, liquid/liquid extraction methods adapted from Davoli (1998) and Nystrom (1997) were performed substituting dichloromethane (DCM) for toluene-n-hexane (1:1). Recoveries were not ideal with the analyte either remaining in the aqueous phase or being lost during the evaporation process. The method was altered to utilize acetone to precipitate proteins out of solution with centrifugation to ensure analyte was not lost in the aqueous phase.

26 The sample was then evaporated to dryness and then reconstituted in DCM. Data produced from samples extracted with acetone and reconstituted with DCM are shown in chapter 3 (Figure 3.3) and chapter 4 (Figures 4.1 and 4.2). Analyte recovery was consistent and clearly showed linear production of cotinine over time suggesting an adequate extraction method.

In attempts to further increase analyte recovery, nicotine, cotinine and cotinine d3 were added to inactive S9 fractions in solvent. The extraction procedure was performed with acetone or ACN in the same manner as above. The extraction performed with

ACN (Figure 7) shows cotinine recovery to be greater than 85% with less variability than the extraction performed with acetone making ACN the preferred solvent for subsequent extractions. Tetrohydrofuran (THF) was added to ACN at 20% as recommended by a protocol created by Dow Chemical (Midland, MI). Data produced from samples extracted with the combination ACN:THF (80:20) is shown in Figure 3.2 (Chapter 3).

Data was consistent and showed linear cotinine production over time suggesting the

ACN:THF combination may be more efficient than ACN alone.

Due to less than ideal recoveries of nicotine, most results from this study are reported in cotinine production. Figure 3.1 (Chapter 3) is the exception, with data reported in loss of nicotine due to insufficient detection of cotinine production.

Subsequent samples were further concentrated prior to GC/MS analysis resulting in greater cotinine detection.

27 CHAPTER 3

NICOTINE METABOLISM IN S9 FRACTIONS

3.1 Introduction

Assays utilizing subcellular fractions are valuable in determining rates of metabolism and/or metabolites produced from exogenous and endogenous compounds.

In vitro assays have the benefit of producing data at the enzymatic level without interference from a complete biological system. In vitro assays are generally inexpensive and much less invasive than in vivo studies. The need for in vitro studies can, in many instances, be predicated or ruled out based on the results of previous in vitro screening techniques (Liu and Jia, 2007; U.S FDA, 1997). This type of data is

regularly used to determine possible drug interactions that can lead to potentially life

threatening scenarios. A decrease in efficacy of pharmaceuticals can be equally as

disastrous as an overdose.

The cytochrome (CYP) 450 proteins contained in S9 and microsomal fractions

possess a heme cofactor group that makes them optically active at 450 nm. It is this

optical quality for which they are named. CYP enzymes catalyze oxidation reactions,

usually as part of an electron transfer chain (Parkinson, 2001). CYP proteins have been

found in almost all biological life forms and some homology between species has been

observed (Raunio et al., 2008). CYP2A6 has been greatly studied and many genetic

polymorphisms have been isolated, many with regards to nicotine metabolism in

humans (Fukami et al., 2005 a,b; Yamano et al., 1990; Oscarson et al., 1999a;

Kitagawa et al., 2001; Ariyoshi et al., 2001; Pitarque et al., 2001; Yoshida et al., 2002;

Daigo et al., 2002; Oscarson et al., 2002; Fukami et al., 2004; Loriot et al., 2001; Schulz

28 et al., 2001; Schoedel et al., 2004; Nakajima et al., 1996; Oscarson et al., 1999b;

Nunoya et al., 1999a,b; Miyamoto et al., 1999; Minematsu et al., 2003; Fujieda et al.,

2004 Chougnet et al., 2009). A number of compounds, in addition to nicotine, are

metabolized to some degree by CYP2A6 in humans and are shown in Table 3.1.

Substrate Assay/end-point Substrate Assay/end-point Coumarin 7-hydroxylation Nicotine N-1′-oxidation Methoxyflurane Dehalogenation Cotinine 3′-hydroxylation Halothane Reduction NNK Mutagenicity 4-methylnitrosamino-1- (3-pyridyl)-1-butanone SM-12502 S-oxidation NDEA Mutagenicity 3,5-dimethyl-2-(3- N-nitrosodiethylamine pyridyl)thiazolidin-4-one hydrochloride Losigamone Oxidation AFB1 Mutagenicity aflatoxin B1 Valproic acid Oxidation MOCA N-oxidation 4,4′-methylene-bis(2- chloroaniline) Letrozole Oxidation 1,3-butadiene Monoxide formation

Disulfiram Sulfoxidation Quinoline 1-oxidation

(l)-Menthol (-)-(1R,3R,4R)-trans- DCBN Protein adduct formation p-menthane-3,8-diol 2,6- dichlorobenzonitrile MTBE O-demethylation methyl tert-butyl ether

The S9 fraction is named based on the process of isolation. Tissue is

homogenized in a biological buffer [1:2] and centrifuged at 9000 g for 20 minutes to

separate the metabolically active components from the fractured cellular endoplasmic

reticulum. The resulting supernatant contains the microsomes and cytosol responsible for phase I and phase II metabolism, respectively. Variations in isolation methods allow

for the homogenate to be centrifuged at a higher speed or for a longer time in order to

29 ‘clean up’ the supernatant but the active material isolated remains the same (Hakura et al., 2001; Connors et al., 2013).

Isolating the microsomal fraction requires a much more extensive centrifugation

process. Minor differences in centrifugation speed, homogenation techniques and/or

buffers have been reported in literature; however, the overall methods and end results

are similar (Boobis et al., 1980; Raucy and Lasker, 1991; Guengerich, 1994; Nelson et

al., 2001; Damre et al., 2009). Traditionally, homogenate prepared as above, is

centrifuged at 10,000 g for 20 minutes; and, after the initial centrifugation, the

supernatant is removed, placed into a new chamber and further centrifuged at 100,000

g for 60 minutes (Nelson et al., 2001; Damre et al., 2009). Due to the increased

concentration of enzymes in microsomal fractions, detection of phase I metabolism is

greatly enhanced over S9 fractions. However, microsomes alone are not suitable to

detect metabolites produced during phase II metabolism (U.S. FDA, 1997; Bjornsson et

al., 2003).

Phase I metabolism includes hydrolysis, reduction and oxidation reactions that

create or expose a functional group on the compound intended for biotransformation.

Some metabolites, such as cotinine, are ready for excretion without further

transformation. Others require the addition of a larger molecular group in order to

facilitate excretion. Phase II reactions, such as glucuronidation, methylation,

acetylation, sulfation or other conjugation options, tend to greatly increase hydrophilicity

allowing for more effective excretion. Phase II reactions are not always dependent on

phase I preparation (Parkinson, 2001).

30 CYP2A6 is the enzyme primarily responsible for nicotine metabolism in humans.

A homologous enzyme, CYP2A5, has been identified in mice suggesting that any

effects (i.e. decreased cotinine production) produced with the addition of menthol to

nicotine would be comparable between human and mouse S9 assays (Witschi et al.,

2005; Raunio, 2008). Rats do not share the CYP2A6/CYP2A5 enzyme and were not

expected to produce significant amounts of cotinine as compared to humans and mice

over similar time points (Nakayama et al., 1993).

Examination of the P450 system in trout has led to the identification of many CYP

genes and associated proteins similar to humans and rodent models (Buhler et al.,

1998; Connors et al., 2013). It is also widely accepted that similarities in nicotinic

receptor structure and function exist between humans and teleosts (i.e. zebrafish)

leading to the assumption that conservation of structure and function is also extended

between humans and trout (Papke et al., 2012; Ninkovic & Bally-Cuif, 2006; Bencan &

Levin, 2008; Eddins et al., 2009; Klee et al., 2011; Levin et al., 2006; Svoboda et al.,

2002). It was unknown if trout S9 fractions exhibit the capacity to transform nicotine into cotinine in a manner similar to humans and rodent models.

The objectives of this project were to: 1) determine similarities in nicotine metabolism between humans and mouse, rat and trout, and (2) determine if the addition of menthol affects nicotine metabolism in humans and other species that do effectively metabolize nicotine to cotinine in a manner similar to humans.

31 3.2 Materials and Methods

3.2.1 Chemicals

(+/-) Nicotine (PS-85, lot 404-60A) and (N-12655) was purchased from

ChemService (West Chester, PA). (+/-) nicotine d3 (N412425, lot 2-FWD-101-2) and

(+/-) cotinine-methyl-d3 (C725005, lot 3-MDB-120-3) was purchased from Toronto

Research Chemical Inc. (TRC, Toronto, Ontario, Canada). (-)-l-menthol (W266523), cotinine (C-061M8703), heparin (H4784), NADPH (N5130), phosphate buffered saline

(PBS) (P5368), and ethyl 3-animobenzoate methanesulfonate salt (MS-222) (A5040) were purchased from Sigma-Aldrich Corporation (USA). TRIS-hydrochloride (BP1756), acetone, dichloromethane (DCM), ethanol and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Houston, Texas).

3.2.2 S9 Fractions

Uniduced mouse S9 fractions (Figure 3.3, A-E) purchased from Celsis

(Baltimore, MD) were obtained from a pool of 157 male ICR/CD-1 mice with a protein content of approximately 20 mg/ml. Uniduced pooled male Sprague-Dawley 344 rat S9 was purchased from Moltox (Boone, NC) with a protein content minimum of 20 mg/ml.

Human S9 fractions purchased from Xenotech (Lenexa, KS) were obtained from a mixed gender pool of 50 individuals with a protein content minimum of 20 mg/ml.

Human S9 fractions purchased from Moltox (Boone, NC) were obtained from a pool of 5 males with a protein content minimum of 20 mg/ml. Live Rainbow trout (10, male and female) were purchased from Pond King (Gainsville, TX). Trout were bagged and transported from Pond King to the laboratory in a cooler surrounded with ice. Animals

32 were euthanized with MS-222 buffered with sodium bicarbonate (1:1) at 300-400 mg/L

each in dechlorinated tap water (UNT Animal Use Application #0702). Livers were

removed, pooled and homogenized in phosphate buffer solution (PBS) at pH 7.4 at a

ratio of 1:2 (tissue:PBS). Homogenate was then centrifuged at 9000 g for 20 minutes at

4ºC to isolate the S9 fraction. The S9 (supernatant) was then removed from the pellet,

placed into micro-centrifuge tubes and stored at -80ºC until needed.

Protein content was verified with the Bradford assay for each fraction before use.

3.2.3 Assay and Sample Preparation

All S9 assays were prepared in three separate volumes that when added

together produced a final concentration of 0.5 mg/ml protein, 1mM NADPH and 10 ppm

nicotine with or without varying menthol concentrations. Total volume for each assay

was adequate to allow two replicate aliquots of 300 µls each to be removed at each time

point. Protein volumes were diluted in TRIS buffer (Figure 3.3, A-E) or PBS (Figures

3.1 and 3.2) at pH 7.4. NADPH was prepared with TRIS buffer (Figure 3.3, A-E) or PBS

(Figures 3.1 and 3.2) at pH 7.4. Nicotine and nicotine plus menthol volumes were

diluted at <1% solvent in DMSO and brought to volume in TRIS buffer (Figure 3.3, A-E)

or PBS (Figures 3.1 and 3.2) at pH 7.4. All reaction mixes were prepared in glass tubes

with the protein volume added at time 0 to begin the reaction. Assays were run in a

water bath (ThermoScientific, 2870) at 37ºC (12ºC trout) with two 300 µl aliquots

removed from each tube at each time point. Samples were placed into fresh glass

tubes with 300 µls cold acetone (Figures 3.1 and 3.3, A-E) or ACN:THF (80:20) (Figure

3.2) to terminate the reaction. Samples were then spiked with cotinine d3 (10 ppm final

33 concentration) as the internal standard and centrifuged for 10-20 minutes at 2000 g to pellet. Supernatant was removed and placed into fresh amber vials. Pellets were reconstituted with 500 µls of cold acetone (Figures 3.1 and 3.3, A-E) or ACN:THF

(80:20) (Figure 3.2) to rinse pellets and again centrifuged as above with the supernatant removed and placed into corresponding vial. Rinse was performed two times. Samples were then evaporated to dryness under a gentle steam of nitrogen in a RapidVap

(LabConoco). Dried samples were reconstituted and rinsed with three replicate 500 µl

aliquots of dichloromethane (DCM) and placed into 2 ml amber vials (Thermo Fisher

Scientific, USA). Samples were then concentrated by evaporating to dryness, re-

suspended in DCM and placed in 50 µl glass inserts (Grace Davison Discovery

Sciences, USA) inside 2 ml amber vials for GC/MS analysis.

*Samples from data shown in Figure 3.2 were filtered through glass pipettes

containing approximately 2-2.5 g sodium sulfate post centrifugation to remove excess

water prior to drying under nitrogen.

**Samples from data shown in Figure 3.3, A-E were filtered through Durapore

PVDF syringe tip filters (EMD Millipore, USA) post reconstitution in DCM and prior to

final concentration under nitrogen and re-suspension in the final concentration for

GC/MS analysis.

3.3 Results

3.3.1 Human, Rat and Trout

Metabolic transformation of nicotine was compared between human, rat and trout

liver S9 fractions (Figure 3.1). As expected, nicotine recovery from the human S9

34 clearly showed a significant (p ≤ 0.05) decrease in nicotine over time. No decrease in nicotine was found in rat or trout S9 at the end of 60 minutes.

15 Human Trout 10 Rat

5 *

0 Nicotine ConcentrationNicotine (ppm) 5 15 30 60 Time (minutes)

3.3.2 Human S9 Assay

An assay utilizing pooled human liver S9 fractions was performed to determine the effects of menthol on nicotine metabolism over time. Cotinine production was significantly (p ≤ 0.05) decreased at the 30-minute and 60-minute time points with the addition of menthol (1 ppm) as compared to the nicotine-only dose (Figure 3.2).

Significant (p ≤ 0.05) increases in cotinine production were observed from the 5-minute to 15-minute and 15-minute to 60-minute time points in the nicotine only dose. Due to loss of a replicate sample at 30-minutes in the nicotine-only dose, statistical analysis at the 30-minute time point was unobtainable. Significant (p ≤ 0.05) increases in cotinine production were observed from the 15-minute to 30-minute and the 30-minute to 60- minute time points in the nicotine plus menthol dose.

0.20 c Nicotine 10 ppm Nicotine + Menthol 1 ppm 0.15 +

0.10 b c a * 0.05 a b a a * a 0.00 Cotinine ConcentrationCotinine (ppm) 0 5 15 30 60 Time (minutes)

3.3.3 Mouse S9 Assay

An assay utilizing pooled mouse S9 was performed to determine effects of menthol on the metabolism of nicotine over time. Data shows significant (p ≤ 0.05) inhibition of cotinine production, as compared with the 10 ppm nicotine control, with the addition of menthol concentrations at 1 ppm, 0.5 ppm and 0.25 ppm (Figure 3.3, A, B and C). Significant (p ≤ 0.05) inhibition was observed at the 30-minute and 60-minute time points with the exception of the 0.25 menthol concentration that did not show significance at the 60-minute time point (Figure 3.3, A, B and C). A missing replicate value in the 0.25 ppm group at the 60-minute time point was not compatible with the statistical analysis method used (Figure 3.3-C). The addition of menthol with nicotine at concentrations of 0.1 ppm and 0.01 ppm did appear to have an inhibitory effect on

36 cotinine production over time; however, neither was considered significant (Figure 3.3,

D and E).

A significant (p ≤ 0.05) effect of time with regards to increased cotinine production was seen between the 5-minute and 30-minute time points in the nicotine-only dose

(Figure 3.3, A-E). Graphs from the menthol at 1 ppm and 0.01 ppm show a significant

(p ≤ 0.05) effect of time on cotinine production between the 30-minute and 60-minute

time points (Figure 3.3, A and E). A significant (p ≤ 0.05) effect of time was seen in the

data between the 5-minute, 30-minute and 60-minute time points with the addition of

menthol at 0.5 ppm (Figure 3.3-B). Data from the menthol dose at 0.1 ppm showed a

significant (p ≤ 0.05) effect of time between the 5-minute and 30-minute time points

(Figure 3.3-D).

A 1.5 b Nicotine 10ppm Nicotine+Menthol 1ppm b 1.0

0.5 a

b a a * 0.0 * Cotinine ConcentrationCotinine (ppm) 5 30 60 Time (minutes)

37 B 1.5 b Nicotine 10ppm Nicotine+Menthol 0.5ppm b 1.0

0.5 a c * b a 0.0 * Cotinine ConcentrationCotinine (ppm) 5 30 60 Time (minutes)

C 1.5 b Nicotine 10ppm Nicotine+Menthol 0.25ppm b 1.0

0.5 a

0.0 * Cotinine ConcentrationCotinine (ppm) 5 30 60 Time (minutes)

D 1.5 b Nicotine 10ppm Nicotine+Menthol 0.1ppm b 1.0

b a 0.5 b

a 0.0 Cotinine ConcentrationCotinine (ppm) 5 30 60 Time (minutes)

38 E 1.5 b Nicotine 10ppm Nicotine+Menthol 0.01ppm b 1.0

b 0.5 a a

a 0.0 Cotinine ConcentrationCotinine (ppm) 5 30 60 Time (minutes)

3.3.4 Statistical Analysis

Data were graphed and analyzed with GraphPad Prism 5 software (La Jolla, CA).

Two-way ANOVAs were used to determine significance (p ≤ 0.05) between groups and

the Bonferroni’s multiple comparison test was used to determine significance (p ≤ 0.05)

between time points. The only exception was data from the human, trout and rat assay

(Figure 3.2) that was analyzed with Tukey’s multiple test comparison.

3.4 Discussion

Nicotine metabolism was assessed between human, mouse, rat and trout S9 fractions. Data from the mouse assay (Figure 3.3, A-E) clearly shows an increase in cotinine production, with the addition of nicotine, over time. Human (Figure 3.2) and

39 mouse (Figure 3.3, A-E) data both show significant (p ≤ 0.05) increases in cotinine production between the 5-minute and 60-minute time points with the nicotine-only dose.

This is consistent with published literature suggesting that mice are an adequate model

organism for cotinine production with regards to human nicotine metabolism (Witschi et

al., 2005; Raunio, 2008).

Due to less than ideal analyte recoveries and the lack of sensitivity of the GC/MS

instrument used for analysis, data shown in Figure 3.1 is reported in loss of nicotine

rather than cotinine production. Relative comparisons can still be made based on the

loss of nicotine over time (Figure 3.1) as this loss corresponds well with the increase in

cotinine production over time shown in Figure 3.2. The inverse relationship of nicotine

loss to cotinine production is also consistent with findings reported in published

literature (Benowitz, 1996a; Benowitz et al., 1990; Benowitz and Jacob 1994; Hukkanen

et al., 2005; MacDougal et al., 2003). Nicotine loss (Figure 3.1) in the human S9 over time was significant when compared to the rat and trout S9 fractions which showed negligible to no loss. Plasma samples obtained from rats, after nicotine administration, have been shown to contain corresponding levels of cotinine; however, the rate of transformation is much slower in rats than in humans (Abobo et al., 2012). The lack of nicotine loss in the rat S9 fraction over time as compared with human and mice S9 fractions suggests that rat S9 fractions are not ideal for this type (i.e. P450) of nicotine metabolic studies and were discontinued. Trout studies were also discontinued based on the findings of no significant nicotine loss over time (Figure 3.1); and thus concluding, that rat and trout S9 fractions do not metabolize nicotine in a manner similar to humans. Further studies are needed to adequately assess nicotine metabolism in

40 trout. It is possible that trout, like the rat model, exhibit a very slow rate of transformation from nicotine to cotinine and may be adequate models for other types of studies regarding nicotine.

In vitro data from human and mouse S9 assays clearly shows menthol induced inhibition of cotinine production from nicotine (Figures 3.2 and 3.3, A-E). Recently, it has

been shown that CYP2A6 appears to also metabolize menthol (Miyazawa et al., 2011).

Inhibition is likely a result of substrate competition for enzymes. Menthol induced

substrate competition may have implications for other compounds (Table 3)

metabolized, completely or partially, by CYP2A6 enzymes (Raunio et al., 2001).

Data produced from the human S9 assay (Figure 3.2) shows significant (p ≤ 0.05)

inhibition of cotinine production over time with the addition of menthol to nicotine at the

30-minute and 60-minute time points, as compared to the nicotine-only dose.

MacDougal et al. (2003) initially reported that menthol inhibits the formation of cotinine

in human microsomes which make up a substantial portion of S9 fractions. Data shown

in Figure 3.2 is consistent with MacDougal’s findings. Decreased cotinine production

with the addition of menthol is also consistent with previous findings observed by

Benowitz et al. (2004) in human smokers of mentholated cigarettes. It should be noted

that in many studies involving repeated doses of nicotine, (i.e. cigarette smokers and

repeated-dose scenarios in model animals) menthol related inhibition of nicotine

metabolism has not been found (Abobo et al., 2012; Clark et al., 1996; Richardson,

1997; U.S. Department of Health and Human Services, 1998; Wagenknecht et al.,

1990; English et al., 1994; Eskenazi and Bergmann, 1995; Caraballo et al., 1998;

41 Perez-Stable et al., 1998). Discrepancies between previous findings may be a result of single versus repeated dosing scenarios and is further discussed in chapter 4.

Menthol related inhibition of cotinine production over time was also seen with mouse S9 fractions (Figure 3.3, A-E). Significant (p ≤ 0.05) inhibition of cotinine production over time was seen at menthol doses of 1 ppm and 0.5 ppm (Figure 3.3, A and B) at the 30-minute and 60-minute time points and at a menthol dose of 0.25 ppm at the 30-minute time point (Figure 3.3-C). Menthol doses of 0.1 ppm and 0.01 ppm did appear to inhibit cotinine production over time (Figure 3.3, D and E); however, observed inhibition was not significant. Although extrapolation of data from one species to another is risky, mice appear to be a suitable model for the study of menthol inhibition of cotinine production with regards to humans (Raunio, 2008). Data from the mouse S9 fractions (Figure 3.3, A-E) show trends of menthol induced deceased cotinine production similar to data produced from human S9 fractions (Figure 3.2). A range of menthol concentrations was used in the current project with consideration to varying values of menthol content in cigarettes between mentholated brands (Celebucki et al.,

2005; Gordon et al., 2011). It is possible that conflicting findings regarding cotinine levels found in menthol smokers could be due, in part, to varying levels of menthol contained in smokers’ cigarettes of choice (Clark et al,. 1996; Benowitz et al., 2004).

Mice, (Chapter 4), dosed with a high (1 mg/kg) or low (0.1 mg/kg) menthol concentration exhibited opposing effects on cotinine production when administered repeated doses similar to what would be seen in human smokers. Increased or decreased cotinine production appeared to be based on the menthol concentration of the repeated dose.

42 Menthol concentrations administered to the mice (Chapter 4) were adjusted based on the data produced from the mouse S9 assays (Figure 3.3, A-E).

In vitro data produced in the current project (Figures 3.2 and 3.3, A-E) provides further evidence suggesting that menthol inhibits nicotine metabolism at the cellular level. A significant (p ≤ 0.05) decrease in cotinine production with the addition of

menthol to nicotine provided justification in the need for further in vivo studies. Results

from the in vivo study are discussed in the following chapter.

43 CHAPTER 4

NICOTINE METABOLISM IN THE MOUSE MODEL

4.1 Introduction

In vitro studies have shown menthol to inhibit human microsomal transformation of nicotine into cotinine (MacDougal et al., 2003). Data shown in chapter 3 (Figures 3.2 and 3.3, A-E) provides more evidence supporting findings from previous studies.

Assays performed with human (Figure 3.2) and mouse (Figure 3.3, A-E) S9 fractions

containing microsomes showed significant inhibition of nicotine metabolism with the

addition of menthol. Findings from in vitro methods may isolate or confirm specific

mechanisms but they cannot realistically show effects within the whole animal. For the

sole purpose of metabolic interaction, mice were chosen for the in vivo portion of this

study. Mice administered a dose of nicotine, as previously shown, appear to produce

cotinine in a manner similar to humans (Raunio et al., 2008). Mice differ in overall

metabolic rate and are not dosed in a manner that represents actual human cigarette

smoking but mice have been shown to be an adequate model for this process (Raunio

et al., 2008). It can be extrapolated that any observed effects in mice produced by the

co-administration of menthol plus nicotine will also be observed in humans under the

same controlled conditions.

Whole animal models are necessary for identification of systemic responses.

While human subjects are regularly used and assessed in cigarette smoking research,

many factors cannot be controlled, leading to sometimes inconsistent findings

(Ahijevych and Parsley, 1999). Measurable parameters are also limited when using

human subjects as compared to using animal models. Many methods are used when

44 attempting to replicate the effects of human smoking in animals, such as utilizing actual cigarette smoke, concentrating cigarette smoke obtained from artificial smoking machines, or individualizing components of cigarette smoke. Common methods of delivery include inhalation (direct or whole body), injection (sub-cutaneous, intravenous, inter-peritoneal), dermal, and oral. These methods are equally valuable; however, none can adequately represent the active inhalation process performed by humans (Hecht,

2005). Inhalation studies utilizing models such as: dogs, hamsters, mice, rats, ferrets,

rabbits and non-human primates have been published assessing various parameters

with mixed results with regards to pulmonary tumor induction (IARC, 2004; Coggins,

1998, 2001 and 2002; Dontenwill et al., 1973; Mauderly et al., 2004; Hutt et al., 2005;

Witschi et al., 2005; Vinegar et al., 1985). Findings from multiple studies suggest that mice may be a more valuable model, with regards to nicotine metabolism, in assessing risk effects in a complete biological system (Hutt et al., 2005; Witschi et al., 2005;

Raunio, 2008). Several strains of mice readily grow tumors when exposed to cigarette smoke and the presence of the CYP2A5 gene suggests that they will metabolize nicotine in a manner similar to humans (Hutt et al., 2005; Witschi et al., 2005; Raunio,

2008).

Studies involving human subjects have reported that African-American smokers of mentholated cigarettes metabolize nicotine more slowly than smokers of non- mentholated cigarettes (Benowitz et al., 2004). African-American smokers tend to smoke fewer cigarettes per day while inhaling higher levels of nicotine and smoke components than Caucasians (Benowitz et al., 2009; Carabello, 1998; Perez-Stable et al., 1998). Some studies have also reported decreased smoking cessation rates in

45 smokers of mentholated cigarettes compared to non-mentholated smokers (Foulds,

2006; Gandhi et al., 2009; Okuyemi et al., 2003, 2007 and 2012). Demographic data

consistently shows that the risk of cigarette-related cancers and disease complications

are more prevalent in African-American smokers when compared with Caucasians

(Harris et al., 1993; MMWR, 2008; Werley et al., 2007). Based on these findings, it was

hypothesized that the addition of menthol may contribute to the increased risk of

disease in African-American individuals (Cummings et al., 1987; Novotny et al., 1988;

Sydney et al., 1989, 1995).

Menthol has been shown to decrease irritation and may also enhance absorption

of components contained in cigarette smoke (Dessirier et al., 2001; Willis et al., 2011).

Menthol has been shown to have a negative effect on nAChRs by allosteric modulation

leading to a decrease in the efficacy of nicotine at the receptor site (Hans et al., 2012).

Increased levels of circulating nicotine suggests a mechanism for the increased density

of nAChRs seen in brain imaging of smokers of mentholated cigarettes possibly

affecting the rate of smoking cessation in individuals attempting to quit (Brody, et al.,

2013). Buproprion is an atypical anti-depressant commonly used as an aid in smoking cessation. It has been suggested that bupropion metabolism may be affected by menthol leading to lower cessation rates in smokers of mentholated cigarettes

(Okuyemi et al., 2003 and 2012). Although findings were not significant with regards to menthol inhibition of bupropion metabolism, there were findings of a small significant increase in bupropion levels in a select group of menthol smokers after cessation

(Okuyemi at al., 2013).

46 The main objectives of the present project were to: 1) determine if menthol inhibits the production of cotinine in the mouse model after a single dose, (2) determine if the addition of menthol inhibits the production of cotinine in the mouse model after a series of repeated doses and (3) determine at which concentration of menthol an effect may be seen.

4.2 Materials and Methods

4.2.1 Model Animals

Male Swiss Webster mice were purchased from Charles River Laboratories and housed in the UNT animal care facility according to protocol. Animals were housed 4 per cage unless separation became necessary due to fighting. Mice were free fed commercially prepared rodent blocks with water available at all times and maintained on

a light/dark cycle of 12/12. Animals were allowed to acclimate for approximately one week before any procedures. Mice were approximately 5-6 weeks old with an average weight of 25 g at the time of procedure. All animal husbandry and methodologies were approved by the UNT Animal Care and Use Committee prior to mice arriving on site

(UNT Animal Use Application # 0807).

4.2.2 Chemicals

(+/-) Nicotine (PS-85, lot 404-60A) was purchased from Chem Service (West

Chester, PA). (+/-) nicotine d3 (N412425, lot 2-FWD-101-2) and (+/-) cotinine-methyl- d3 (C725005, lot 3-MDB-120-3) was purchased from Toronto Research Chemical Inc.

(TRC, Toronto, Ontario, Canada). (-)-l-menthol (W266523), cotinine (C-061M8703),

47 heparin (H4784) and phosphate buffered saline (P5368), were purchased from Sigma-

Aldrich Corporation (USA). TRIS-hydrochloride (BP1756), acetone, dichloromethane, ethanol and DMSO were purchased from Fisher Scientific (Houston, Texas).

4.2.3 Acute Single-Dose

Two mice per time point were administered a single dose at either: 1mg/kg

nicotine, 1mg/kg nicotine + 0.5 mg/kg or 1 mg/kg nicotine + 0.01 mg/kg menthol.

Injection solutions were created from stocks of nicotine (1500 ppm) and menthol (150

ppm and 3 ppm). Total injection volume was 200 µls made up of nicotine solution in not

more than 1% ethanol carried in phosphate buffered saline (PBS). Dose was

administered via a single sub-cutaneous injection using insulin needles with 1cc

syringes (26g BD). Two mice per time point for each dose were anesthetized in a

chloroform chamber and blood was drawn via cardiac puncture with heparinized 26g

needles and 3cc syringes (BD) before termination via cervical dislocation. Whole blood

was placed into heparinized micro-centrifuge tubes and centrifuged with a small

portable centrifuge until separation. Plasma was then placed into fresh micro-centrifuge

tubes and stored at -80ºC until extraction. Mice were sacrificed at 15, 30, 60 and 120

minutes post injection.

4.2.4 Repeated Dose 14-day

Mice were administered a single sub-cutaneous dose every day for 14 days at

concentrations indicated above. On day 14, after injections, mice were terminally

48 sampled as described above. Mice were sacrificed at 15, 30, 60, 120 and 180 minutes after final injection.

4.2.5 Sample Prep

Plasma was obtained by centrifugation and stored at -80ºC until extraction. After thawing, 200 µls of plasma were spiked with cotinine d3 for a final concentration of 10 ppm as internal standard and placed into glass tubes. Plasma was washed two different times with 5 mls acetone, vortexed and centrifuged at 2000 g for 10 min to pellet. Supernatant from both washings was removed and placed together into a fresh glass vial and evaporated to dryness with a gentle stream of nitrogen in a RapidVap

(LabConoco). Dried samples were reconstituted and rinsed with three replicate 500 µl aliquots of dichloromethane (DCM) and filtered through Durapore PVDF syringe tip filters (EMD Millipore, USA). Filtered samples were placed into 2 ml amber vials

(Thermo Fisher Scientific, USA), evaporated to dryness and re-suspended in 40 µls

DCM in 50 µl glass inserts (Grace Davison Discovery Sciences, USA) for GC/MS analysis.

4.3 Results

Data (Figure 4.1) indicates inhibition of cotinine production in mice after a single dose with the addition of menthol. A single dose with the addition of menthol (0.5 mg/kg) showed a decrease in cotinine production at all time points as compared to nicotine only. A single dose with the addition of menthol (0.01 mg/kg) was initially higher (15-minute and 30-minute time points) than the nicotine only dose, but over time,

49 showed a decrease in cotinine production (60-minute and 120-minute time points)

similar to the higher menthol dose.

0.3 Nicotine 1 mg/kg Nicotine+Menthol 0.5 mg/kg 0.2 * Nicotine+Menthol 0.01 mg/kg

0.1

0.0 15 30 60 120 Time (minutes) -0.1 Cotinine ConcentrationCotinine (ppm)

After a 14-day dosing regime (Figure 4.2), cotinine production for all groups is lower at the 15-minute time point than after a single dose. An elevated production of cotinine, relative to the nicotine-only control group, is observed in the repeated high menthol (0.5 mg/kg) group similar to the single low dose menthol (0.01 mg/kg) group

(Figure 4.2) at the 15-minute time point. Elevation of cotinine production in the 14-day

group is consistent for all data points. Cotinine production appears to be inhibited at all

points (Figure 4.2), relative to the nicotine-only control, by a low menthol dose (0.01

mg/kg) in the repeated exposure group.

Cotinine production in the single dose nicotine-only group was substantially

higher than cotinine production in the repeated-exposure nicotine-only group.

50 A significant (p ≤ 0.05) decrease in cotinine production was found between the low menthol (0.01 mg/kg) single exposure and the low menthol (0.01 mg/kg) repeated

14-day exposure.

0.3 Nicotine 1 mg/kg Nicotine+Menthol 0.5 mg/kg 0.2 Nicotine+Menthol 0.01 mg/kg

0.1

0.0 * Cotinine ConcentrationCotinine (ppm) 15 30 60 120 180 Time (minutes)

4.4 Statistical Analysis

Data were graphed and analyzed with GraphPad Prism 5 software (La Jolla, CA).

Two-way ANOVAs were used to determine significance (p ≤ 0.05) between groups and

the Bonferroni’s Multiple Comparison test was used to determine significance (p ≤ 0.05)

between time points.

4.5 Discussion

Mice dosed with 1 mg/kg nicotine co-administered with either 0.5 mg/kg or 0.01

51 mg/kg menthol showed marked differences in cotinine production between timed samples after a single sub-cutaneous dose (Figure 4.1) versus a repeated (14 day) sub- cutaneous dose (Figure 4.2). Differences were found between the nicotine-only groups and each menthol group. Mice administered nicotine-only exhibited average plasma cotinine levels at the 15-minute time point of ~1.4 µg/ml (140 ng/ml) and ~0.8 µg/ml (80 ng/ml) for the acute dose (Figure 4.1) and repeated dose (Figure 4.2), respectively.

This is consistent with plasma cotinine levels in human smokers previously reported at

100-300 ng/ml with typical ranges from 20-60 ng/ml in arterial blood (Armitage et al.,

1975; Henningfield et al., 1993; Gourlay and Benowitz, 1997; Rose et al., 1999; Lunell

et al., 2000; Benowitz et al., 1983a; Gori and Lynch, 1985; Yamazaki et al., 2010).

Plasma cotinine levels in human smokers have been reported at 21-4420 ng/ml with an

average of 379.4 ng/ml (Massadeh et al., 2009). This suggests that the nicotine dose at

1 mg/kg is a relatively low dose when compared to plasma cotinine levels measured in

human smokers. It is likely that the average plasma cotinine values seen in mice in the current project (Figures 4.1 and 4.2) have been under-represented due to low analyte recovery as discussed in chapter 2. In the case of under-representation, cotinine plasma values in the current project would still fall within reported ranges (Massadeh et al., 2009).

Mice dosed with only nicotine exhibited lower levels of plasma cotinine after the repeated dosing regime than with a single-dose administration (Figures 4.1 and 4.2).

Although findings did not show significance between the two groups when analyzed with

GraphPad Prism, it should be noted that previous analysis using SPSS (v. 13.0; SPSS,

52 Chicago, IL, USA) did show significant findings at times 30, 60 and 120 minutes

(Petersen et al., unpublished).

A previous study by Schodel et al. (2003) performed a 21-day repeated dosing

regime of nicotine in monkeys. Data showed a decrease in CYP2A6 activity after

repeated nicotine exposure. The author suggests that down-regulation of CYP2A6

associated mRNA may be responsible for this decrease. Extrapolating this data to the

mouse model, it is possible that CYP2A5 activity is also decreased in the mouse model

after repeated exposure to nicotine. This may be responsible for the lower production of

cotinine seen in the repeated dosing schedule (Figure 4.2) as compared to the single

dose (Figure 4.1) observed in mice who received only nicotine (Schodel et al., 2003).

This effect was not seen between a single and repeated nicotine dose scenario in rats

exposed to cigarette smoke via inhalation. After a repeated dose, cotinine production

(Cmax) in rats appeared to almost double in concentration as compared to the single

dose (Abobo et al., 2012). This discrepancy may be a result of species specific

metabolism, route of exposure, or any number of interactions between compounds in

cigarette smoke.

The graph from the single-dose administration (Figure 4.1) shows decreased

cotinine production with the addition of 0.5 mg/kg menthol when compared with the

nicotine-only dose. The lower menthol dose (0.01 mg/kg), while initially high, also

appears to inhibit cotinine production over time, although not as substantially as the

higher dose. These findings were not unexpected and are consistent with the present in

vitro data (Chapter 3, Figures 3.2 and 3.3). Data is also consistent with previously

published literature regarding inhibition of nicotine metabolism in human microsomes

53 and individuals with the addition of menthol (MacDougal et al., 2003; Benowitz et al.,

2004). Although important for determining interactions and relationships, single-dose scenarios do not effectively replicate human smoking habits.

The graph from the repeated-dose regime (Figure 4.2) shows an increase in cotinine production with the addition of menthol (0.5 mg/kg) as compared to the nicotine-only dose. This suggests that the higher menthol dose may be sufficient to up- regulate CYP2A6 enzyme action creating a more rapid cotinine formation.

The recent study by Abobo et al. (2012) produced findings that are similar to data shown in the present project. Rat groups were exposed to either mentholated or non- mentholated mainstream cigarette smoke as a single dose or a series of repeated doses. The repeated-dose regime was performed every 12 hours for the duration of 17 cigarettes at 10 puffs per exposure. Three major differences existed between the present study and the study performed by Abobo et al.: 1) model organisms were rats,

2) cigarette smoke was administered via inhalation and 3) plasma levels of nicotine, in addition to cotinine, were assessed. In this case, extrapolation of data from rats to mice should be adequate when differing rates of metabolism between species are considered

(i.e., time).

Data from the single-dose rat group was consistent with the current mouse single-dose data showing a decrease in cotinine production when exposed to menthol cigarette smoke as compared to the non-mentholated cigarette smoke (Figure 4.1).

One benefit to the Abobo et al. (2012) study was the assessment of nicotine levels concurrently with cotinine. Nicotine absorption (Cmax) was substantially, although not statistically significantly, less in those exposed to mentholated smoke as compared to

54 non-mentholated smoke after the single and after repeated dosing regimes. The author suggests that menthol may inhibit absorption of nicotine. The present study was not able to assess plasma nicotine levels; however, both groups of mice did receive an equal dose of nicotine via sub-cutaneous injections. This suggests that menthol inhibition of nicotine absorption was not the cause of lowered cotinine production found in the mice.

Rat data from the repeated cigarette smoke exposure also showed similar findings as the present study. Rats exposed to mentholated smoke exhibited a longer mean elimination half-life for nicotine than rats exposed to non-mentholated smoke.

Cotinine production (Figure 4.2) in mice resulting from the addition of menthol at 0.5

mg/kg is consistent with an increase in metabolic transformation from nicotine to

cotinine suggested by Abobo et al. (2012). Mice dosed with menthol at 0.01 mg/kg did

not show this increase in cotinine production (Figure 4.2) which suggests a substantial

concentration of menthol is needed to enact this change in metabolism.

The increased cotinine production observed after the repeated high dose

menthol (0.5 mg/kg) in the present study (Figure 4.2) is also consistent with the findings of Clark et al. (1996). Numerous others have also reported increased cotinine levels in

African American smokers (Richardson, 1997; U.S. Department of Health and Human

Services, 1998; Wagenknecht et al., 1990; English et al., 1994; Eskenazi and

Bergmann, 1995; Caraballo et al., 1998; Perez-Stable et al., 1998). The Clark study

comprised of 161 smokers showed increased levels of cotinine in menthol smokers as

compared to non-menthol smokers (Clark et al., 1996). This is contrary to the

observations of Benowitz et al. (2004) where menthol appeared to decrease cotinine

55 formation. The in vitro data shown in chapter 3 (Figure 3.3, A-E) and the repeated low

dose (0.01 mg/kg) menthol data (Figure 4.2) are also consistent with the findings of

Benowitz et al. (2004). It is unclear why these inconsistencies exist. Data from animal

studies is difficult to extrapolate to humans due to differences in dose administration,

species-specific metabolic processes, and the fact that human subjects can often be

unreliable when self-reporting.

It has been postulated that menthol may inhibit not only nicotine metabolism, but

also, the metabolism of nicotine metabolites (e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone (NKK)) (Sarkar et al., 2012). Metabolic inhibition of nicotine metabolites (e.g.

cotinine, NKK, etc.) could lead to elevated plasma levels of nicotine metabolites for a

greater period of time possibly increasing risk of cigarette related disease. Elevated

cotinine levels, as discussed earlier, have previously been reported in African-American

menthol smokers (Clark et al., 1996; Richardson, 1997; U.S. Department of Health and

Human Services, 1998; Wagenknecht et al., 1990; English et al., 1994; Eskenazi and

Bergmann, 1995; Caraballo et al., 1998; Perez-Stable et al., 1998). In the study by

Sarkar et al. (2012), glucuronide metabolite ratios were measured daily between adult

menthol and non-menthol smokers. Sarkar (2012) reported no significant differences in ratios of nicotine or NKK to corresponding metabolites in menthol smokers when

compared to non-menthol smokers. In the current study, the repeated high menthol

dose (0.5 mg/kg) in mice (Figure 4.2) clearly shows an elevation in cotinine production

when compared to the nicotine-only dose. It is unknown if this increase is due primarily

to the increase in nicotine transformation or to a decrease in cotinine metabolism.

56 Controversy still exists in determining the effect of menthol on nicotine metabolism. Some studies have reported no significant differences in cotinine levels between mentholated and non-mentholated smokers (Allan and Unger, 2007; Muscat et al., 2009). Most agree that the likelihood of an elevated cancer risk associated with mentholated cigarettes, as compared to non-mentholated, is slim (Benowitz et al., 2004;

Heck, 2010). However, it is still largely unknown what effects menthol may have on other aspects of metabolic interactions or on smoking behaviors (Okuyemi et al., 2012).

The focus of the present study makes these data unique. The effect of menthol on nicotine metabolism was observed in a mouse model that more closely replicates human enzymatic transformation than the traditional rat model. Sub-cutaneous injections ensured that each individual mouse received an equal dose as inhalation administration is typically difficult to regulate. Cotinine production was both decreased

(Figures 4.1 and 4.2) and increased (Figure 4.2) depending on dose and length of exposure. More research is needed to uncover discrepancies between menthol and non-menthol smokers. Based on the idea of the 3-Rs, (i.e. Reduce, Refine and

Replace) current experimental design employed a minimum number of animals.

However, a greater number of animals (e.g. 10 per time point) may be needed to confirm the results of the present study.

57 CHAPTER 5

CONCLUSIONS AND SUMMARY

This study investigated the biotransformation of nicotine in multiple animal models. The three major findings were:

• Mice appear to metabolize nicotine, over time, in a manner similar to humans.

• Menthol decreases cotinine production over time, after a single dose, in mice.

• Menthol increases cotinine production over time, after repeated doses, in mice.

In assessing various species for the study of nicotine metabolism, the mouse model has been shown to more accurately represent human biotransformation from nicotine to cotinine. Menthol does appear to influence the metabolism of nicotine in vitro and in vivo in the mouse model. The effects produced are varied and appear to be dependent on the concentration of menthol administered and whether it is a single exposure or a consistently repeated dose. Initially, a single-dose of menthol with nicotine was shown to inhibit the production of cotinine over time in the in vitro mouse and human S9 fractions (Figures 3.2 and 3.3, A-E) and in the in vivo (Figure 4.1) mouse model. Menthol induced inhibition of cotinine formation from nicotine is consistent with previous findings (MacDougal et al., 2003; Benowitz et al., 2004; Abobo et al., 2012).

However, the high menthol (0.5 mg/kg) repeated-dose produced an increase in cotinine production over time in the in vivo mouse model (Figure 4.2). An increase in cotinine production with the addition of menthol to nicotine was also observed in the rat model after repeated dosing (Abobo et al., 2012).

Controversy still exists with regards to the higher risk of disease associated with mentholated cigarettes. The bulk of published evidence does not support an increased

58 risk of cancer due to mentholated cigarettes as compared to non-mentholated cigarettes

(Heck, 2010). However, menthol has been strongly suggested as a possible catalyst in behaviors associated with smoking and the difficulty associated with smoking cessation

(Okuyemi et al., 2007).

More research is needed to determine the physiological scope of effects that may be produced by menthol. It is unlikely that menthol makes smoking more dangerous; however, it is possible that menthol makes smoking more rewarding. Cigarette smoking is sometimes replaced and/or supplemented with the now popular smokeless cigarettes marketed as the safer alternative. Many smokeless cigarettes are equipped with adjustable nicotine and menthol outputs; however, safety margins of smokeless cigarette use have not been established. It is possible that menthol may also interact with other compounds that are metabolized by CYP2A6 leading to decreased or increased efficacy of pharmaceuticals (Okuyemi et al., 2003, 2012). Menthol is a

G.R.A.S. compound and considered safe according to the FDA. This rating is unlikely to change but further studies could provide evidence that menthol should be used with caution in certain instances.

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