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QUANTITATIVE METABOLIC CLEARANCE OF GLYCOL ETHERS USING CRYOPRESERVED HEPATOCYTES BY FERMIN E. GUERRA A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Katherine Walstrom Sarasota, Florida May, 2012
ii DEDICATION This senior thesis, the culmination of my undergraduate career, is dedicated to my parents Placido Fermin Guerra and Hilda Guerra, and brother, Placido Erick Guerra, for their limitless support and encouragement.
iii ACKNOWLEDGMENTS I would like to thank Dr. Christopher Wegerski and Dr. Robert Rubin of Lovelace Respiratory Research Institute (Albuquerque, NM) for providing me and other New College of Florida students with an invaluable opportunity to co nduct research at Lovelace. I would like to further extend my gratitude to Dr. Christopher Wegerski for his guidance, patience, and positive support throughout this research project, both while I was in Albuquerque conducting research and after my return t o New College. I would also like to thank my sponsor, Dr. Katherine Walstrom, for her positive feedback throughout the process. Dr. Walstrom has also been a great advisor who has guided me during my years at New College and has shown sincere interest in my future endeavors. Finally, I would like to thank Dr. Paul Scudder and Dr. Elzie McCord for taking time to be a part of my committee.
iv Table of Contents Title Page Dedication ii Acknowledgments iii List of Tables vi List of Figures vii Abstra ct ix Chapter 1: Literary Review 1 I. National Toxicology Program 1 II. E nvironmental Protection Agency and Significant New Use Rule 3 A. Glymes 3 III. Glycol Ethers 13 A. E thylene Glycol Monobutyl Ether 14 B. Ethylene Glycol Monohexyl Ether 22 C. Ethylene Glycol 2 Ethylhexyl Ether 26 Chapter 2: Materials & Methods 30 I. High Performance Liquid Chromatography Mass Spectrometry 30 A. Parameters 33 II. Experimental Procedures 36 Chapter 3: Results 43 I. Half life Quantitation 43 II. Metabolites 53
v Table of Content s Chapter 4: Discussion 59 I. Metabolic Clearance 59 II. Cryopreserved Hepatocytes Good Metabolism Models? 62 III. Future Directions and Conclusion 64 Appendix A Paramete rs and Sample Spectra of Effects 66 Appendix B Additional Glycol Ether Information 72 I. Ethylene Glycol Monobutyl Ether 72 References 74
vi List of Tables 1 1. Chemicals Listed in the Proposed Significant new Use Rule 4 1 2. Physical and Chemical Properties of Ethylene Glycol Monobutyl Ether 16 1 3. Physical and Chemical Properties of Ethylene Glycol Monohexyl Ether 23 1 4. Physical and Chemical Properties of Ethylene Glycol 2 Ethylhexyl Ether 27 2 1. Mass of Positive Ions Filtered Through Q1 and Q3 Chambers 41 3 1. Results From the Incubation of Female Human Hepatocytes with Ethylene Glycol 2 Ethylhexyl Ether 44 3 2. Half lives (in minutes) of All Incubations with Ethylene Glycol 2 Ethylhexyl Ether, Ethylene Glyco l Monohexyl Ether, and the Positive Control 7 E thoxycoumarin 46 3 3. Data from the Female Human Hepatocyte Incubations with Ethylene Glycol Mo nobutyl Ether 51 A 1. API 5000 Triple Quadrupole LC/MS/MS Analyte Parameters 71
vii List of Figures 1 1. National Toxicology Program 2 1 2. Monoglyme 6 1 3. Major Metabolic Pathways for Monoglyme 7 1 4. Diglyme 9 1 5. Metabolism Pathway of Diglyme 11 1 6. Effects of Diglyme on Rat Testes 11 1 7. Ethylene Glycol Monobutyl Ether 15 1 8. Metaboli sm Pathway for Ethylene Glycol Monobutyl Ether 18 1 9. Effects of Calcium on Hemolysis and Mean Cell Volume 21 1 10. Synthesis of Ethylene Glycol Monohexyl Ether 23 1 11. Chemical Structure of Ethylene Glycol 2 Ethylhexyl Ether 27 2 1. MS/MS Multiple Reaction Monitoring 31 2 2. Schematic of the Component of an APCI Source 32 2 3. Mechanism of APCI 33 2 4. Effect of Collision Energy on Protonated Ethylene Glycol 2 Ethylhexyl Ether Parent and Fragment Ions 35 2 5. Example Integration of Peaks Using Analyst 1.5 Software 42 3 1. Trial 2 Spectra of Ethylene Glycol 2 Ethylhexyl Ether Incubated with Female Human Hepatocytes 43 3 2. Percent Remaining of Ethylene Glycol 2 Ethylhexyl Ether Incubated With Female Human Hepatocytes 45
viii List of Figures 3 3. Natural Logarithm of the Percent Remaining of Ethylene Glycol 2 Ethylhexyl Ether Incubated with Female Human Hepatocytes 45 3 4. Half lives of All Incubations with Ethylene Glycol 2 Ethylhexyl Ether 48 3 5. Half lives of All Incubations with Ethylene Glycol Monohexyl Ether 49 3 6. Half lives of Female and Male Human and Mouse Hepatocytes with 7 Ethoxycoumarin 50 3 7. Half lives of All Incubations with the Positive Control 7 Ethoxycoumarin 50 3 8. Percent Remaining of Ethylene Glycol Monobu tyl Ether Incubated with Female Human Hepatocytes 52 3 9. Retention Peak of Ethylene Glycol Monobutyl Ether in the No Compound Negative Control of Female Human Hep atocytes 52 3 10. Retention Peak of Ethylene Glycol 2 Ethylhexyl Ether and Suspected Metabolite 54 3 11. Suspected Ethylene Glycol 2 Ethylhexyl Ether Glucuronide Metabolite 55 3 12. Detection Peaks of Ethylene Glycol 2 Ethylhexyl Ether and the Suspected Metabolite 56 3 13. Spectra of Male Mouse Hepatocytes Incubations When Scanning for the Ethylene Glycol 2 Ethlhexyl Ether Glucuronide Metabolite with Q1/Q3 Masses of 351.000/63.000 and a Decl ustering Potential of Zero 57 4 1. Glucuronidation Mechanism of Hydroxyl Groups by UDP glucuronosyl Transferase 60 A 1. Schematic of the API 5000 69 A 2. Fragmentation pattern of ethylene glycol monobutyl ether, and ethylene glycol monohexyl ether when Collision Enery is 15 V 70
ix QUANTITATIVE METABOLIC CLEARANCE OF GLYCOL ETHERS USING CRYOPRESERVED HEPATOCYTES Fermin Ernesto Guerra New College of Florida, 2012 ABSTRACT The National Toxicology Program report s more than 80,000 chemicals registered fo r use in the United States An estimated 2,000 new chemicals are introduced for use in everyday items such as foods, personal care products, prescription drugs, household cleaners, and lawn care products each year. The glycol ethers ethylene glycol monohexyl ether and ethylene glycol 2 ethylhexyl ether lack toxicity and met abolism studie s even though their chemical structure s are similar to ethylene glycol monobutyl ether, a hematotoxin. Cryopreserved hepatocytes from female and male human, mouse, and rats were used to d etermine the half lives of glycol ethers as well as possible metaboli tes The half life of e thylene glycol 2 ethylhexyl ether was shorter than the half life of ethylene glycol monohexyl ether. However, the half life of ethylene glycol monobutyl ether could not be determined possibly due to metabolite interference and detect ion of the compound in the negative control. Also, glucuronidation was observed with ethylene glycol 2 ethylhexyl ether which is the first time a metabolite has been detected for this glycol ether in the literature. Dr. Katherine Walstrom Division of Natu ral Sciences
1 CHAPTER 1 LITERARY REVIEW I. NATIONAL TOXICOLOGY PROGRAM Established in 1978, the National Toxicology Program was created to coordinate toxicology testing programs within the federal government, strengthen the science base in toxicology, develop and validate improved testing methods, and provide information about potentially toxic chemicals to health, regulatory, and research agencies, scientific and medical communities, and the public (National Toxicology Program, 2004) The need for the National Tox icology Program arose because of increasing scientific, regulatory, and Congressional concerns about the human health effects of chemical agents in our environment. Permanent status of the National Toxicology Program was granted in 1981 because many human diseases were thought to be directly or indirectly related to chemical exposures. Therefore, it was thought that decreasing or eliminating human exposures to those chemicals would help prevent some human disease and disability. Three agencies form the core of the National Toxicology Program: 1. National Institute of Environmental Health Sciences of the National Institutes of Health, 2. National Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention, 3. Food and Drug A dministration through its National Center for Toxicological Research (National Toxicology Program, 2012 a ) The diagram shown in Figure 1 1 shows the agencies that comprise the National Toxicology Program. According to National Toxicology Program informati onal website (National Toxicology Program 2005), there are more than 80,000 chemicals registered for use in the United States. An estimated 2,000 new chemicals are introduced for use in everyday
2 items such as foods, personal care products, prescription dr ugs, household cleaners, and lawn care products each year. Although we may be exposed to these chemicals while manufacturing, distributi ng, using, and disposing we do not know the effec ts of many of these chemicals on our health. Moreover, these chemicals can become pollutants in our air, water, or soil. While relatively few chemicals may pose a significant risk to human health, safeguarding public health entails identifying chemical effects and exposure levels that may be hazardous to humans. By developin g and applying tools of modern toxicology and molecular biology, the National Toxicology Program evaluates agents of public health concern. Figure 1 1 National Toxicology Program, NTP. (National Toxicology Program, 2012 a ).
3 II. ENVIRONMENTAL PROTECTION AGENCY AND SIGNIFICANT NEW USE RULE The Environmental Protection Agency is responsible for ensuring that federal laws protecting human health and the environment are enforced fairly and effectively. These laws sometimes regula te some of the 2,000 new chemicals introduced annually or they restrict or remove chemicals that are currently marketed In July 2011, the E nvironmental P rotection A gency released a report proposing a significant new use rule (SNUR) under section 5(a)(2) o f the Toxic Substances Control Act for 14 glymes (Environmental Protection Agency 2011). The proposed SNUR would require persons who plan to manufacture, import, or process these chemicals for an activity that is designated as a signifi cant new use by this proposal must notify the E nvironmental P rotection A gency at least 90 days before commencing that activity. This notification is required to allow the Environmental Protection Agency time to evaluate the intended use and prohibit or limit that activity if necessary. Under the new proposed rule, the Environmental Protection Agency volume of manufacturing and process ing of the chemical substance, the extent to which a use changes the type or form of exposure of humans or the env ironment to the chemical, and the anticipated method of manufacturing, processing, distribution in commerce, and disposal of a chemical substa nce. A. GLYMES The Enviro nmental Protection Agency lists 14 glymes with similar molecular structures, physical and chemical properties, and potential uses in the SNUR. f monoglyme, diglyme, and ethylglyme. Due to the lack of available use, exposure, and
4 toxicity information of the remaining 11 glymes with similar chemical structures the Environmental Protection Agency decided to include them in the new rule. The 14 glym es are listed in Table 1 1 with some of their proposed consumer uses (Environmental Protection Agency 2011). Table 1 1 Chemicals Listed in the Pr oposed Significant New Use Rule (Environmental Protection Agency 2011). Chemical Abstract Service (CAS) Registry Number (CASRN) Chemical Abstract Index Name Common Name Proposed Excluded Consumer Uses 110 71 4 Ethane, 1,2 dimethoxy Monoglyme or Monoethylene glycol dimethyl ether in electrolyte solutions for sealed lithium batteries 111 96 6 o xybis[2 methoxy Diglyme or diethylene glycol dimethyl ether as a solvent in printing inks for consumer products 112 36 7 oxybis[2 ethoxy E thyldiglyme or diethylene glycol diethyl ether 112 49 2 2,5,8,11 Tetraoxadodecane Triglyme or Triethylene glycol dimethyl ether as a solvent in consumer adhesives as a component of consumer brake fluids as a component of consumer paint/graffiti removers in consumer paints 112 73 2 [oxybis(2,1 ethanediyloxy)]bis Butyldiglyme or Diethylene glycol dibutyl ether 112 98 1 5,8,11,14,17 Pentaoxa heneicosane Tetraethylene glycol di but yl ether 143 24 8 2,5,8,11,14 Pentaoxapentadecane Tetraglyme or Tetraethylene glycol dimethyl ether as an HFC/CFC lubricant as a solubilizing agent for consumer printing inks as a coalescing agent in consumer paints 629 14 1 Ethane, 1,2 diethoxy Ethylglyme or Ethylene glycol diethyl ether
5 Table 1 1, continued Chemicals Listed in the Pr oposed Significant New Use Rule (Environmental Protection Agency 2011). Chemical Abstract Service (CAS) Registry Number (CASRN) Chemical Abstract Index Name Common Name Proposed Excluded Consumer Uses 4353 28 0 3,6,9,12,15 Pentaoxaheptadecane Tetraethylene glycol diethyl ether 23601 39 0 3,6,9,12,15,18 Hexaoxaeicosane Pentaethylene glycol diethyl ether 24991 55 7 Poly(oxy 1,2 ethanediyl) .alpha. methyl .omega. methoxy Polyglyme or Polyethylene glycol dimethyl ether use in consumer paint strippers 31885 97 9 Poly(oxy 1,2 ethanediyl), .alpha. butyl .omega. butoxy Polyethylene glycol dibutyl ether 51105 00 1 5,8,11,14,17,20 Hexaoxatetracosane Pentaethylene glycol dibutyl ether 63512 36 7 5,8,11,14 Tetraoxaoctadecane Butyltriglyme or Triethylene glycol dibutyl ether The Environmental Protection Agency acknowledges there are differences in the proposed chemical uses but decided to act on them together due to their similarities. Toxicological data are available for 3 of the 14 chemicals listed in Table 1 1. Furthermore, glycol ethers similar in structure to glymes and may also be regulated by the Environmental Protection Agency. Studies on the glycol ether ethylene glycol monobutyl ether have found tha t it is a hematotoxin. Ethylene glycol monohexyl ether and ethylene glycol 2 ethylhexyl ether have similar structures as ethylene glycol monobutyl ether but have not been extensively studied O nly very limited acute animal toxicity studies could be found i n the literature for ethylene glycol 2 ethylhexyl et her. The lack of ethylene glycol 2 ethylhexyl ether information motivated this study. Ethylene
6 Figure 1 2 Monoglyme (National Toxicology Program, 2012b). glycol monobutyl ether and ethylene glycol monohexyl ether were included in the study because of their simila r structures Monoglyme The Environmental Protection Agency released a report with concerning toxicological data regarding monoglyme, or 1,2 dimethoxyethane, whose chemical structure is shown in Figure 1 2 (Environmental Protection Agency, 2001) Monoglyme is a clear volatile liquid that is miscible with water and most organic solve nts. The physical and chemical properties of the 1,2 dimethoxyethane, the dimethyl ether of ethylene glycol, make it a great solvent for various applications. Monoglyme is used in lithium batteries due to its low viscosity and cation solvating property (Fe rro Corporation 1993). Acute toxicity studies using rats revealed that the acute oral LD 50 was >4000 mg/kg (DOW Chemical USA, 1983; Environmental Protection Agency, 2001). This study was based on four dose s using four animals per group and supported by another industrial toxicology study that reported the oral rat LD 50 to be >3200 mg/kg. A two dose, six hour inhalation study revealed that the rat LC50 was between 20 and 63 mg/L. At high levels, the vapors caused irritation and anesthesia. Although all hi gh dose animals survived the initial exposure, all test subjects died within 72 hours post exposure The specific metabolic pathways for monoglyme are not known; however, the metabolic pathways for this class of chemical are well known. Most toxic effect s of the monoalkyl glycol ethers are due to the metabolic conversion of the glycol ether into a substituted acetic acid derivative. The metabolite 2 methoxyethanol can be converted into methoxy acetic acid, or ethylene glycol which can be subsequently transformed to oxalic
7 acid (Environmental Protection Agency, 2001). A small portion of 2 methoxyethanol can be excreted by the lungs or kidneys before being metabolized (Cheever et al., 1984) The conversion to ethylene glycol is the result of a demethylation reaction by mixed function oxidases which is a slow reaction. On the other hand, dehydrogenase enzymes first convert the free alcohol to an aldehyde and then to the carboxylic acid ( Klassen, 2001). The latter is a rapid conversion and a dose of 2 methoxyethanol affecting embryonic development is completely oxidized in one hour in rats. Comparatively, the pharmokinetics in rats reveal that the ratio of production for 2 methoxyacetic acid:ethylene is about 5:1. Figure 1 3 Major metabolic pathways for monoglyme. Thickness of arrows indicates relatively rate of formation (Environmental Protection Agency, 2001).
8 There is no definitive chemic al evidence for the metabolism of monoglyme to 2 methoxyethanol, but there is strong biological evidence for this conversion. A study with pregnant mice revealed that fetal body weights were significantly reduced in litters treated with monoglyme (Hardin a nd Eisenmann, 1987). Three other glycol ethers were also tested, but only monoglyme show ed this result. In addition, paw defects were observed in 86.7% of monoglyme treated litters and 87.5% of 2 methoxyethane treated litters, although 2 methoxyethane af fected a larger percent of fetuses than monoglyme, 68.5% to 33.8%, respectively. These similarities are thought to be caused by an in vivo conversion to a common toxin, most likely methoxyacetic acid. Furthermore, Tirmen stein (1993) showed in an in vitro s tudy that microsomal cytochrome P 450 oxi dizes diglyme to 2 methoxyethanol and 2 (2 methoxyethoxy)ethanol. Monoglyme and diglyme are very similar in structure and both human and rat liver microsomes catalyzed the oxidation of diglyme. Figure 1 3 shows the expected metabolism of monoglyme. Excess monoglyme is thought to inhibit the conversion of 2 methoxyethane to ethylene glycol which increases the yield of 2 methoxyacetic acid. Monoglyme has been shown to be a genetic and reproductive toxi n. McGregor et al. (1983) used a n Ames test which assesses the mutagenic potential of chemicals, with monoglyme and alcohol dehydrogenase that showed the test chemical to be cytotoxic to Salmonella typhimurium. However, in the same study Unscheduled DNA Syn thesis assays using mammalian rat hepatocytes did not reveal any monoglyme toxicity. Mouse sperm abnormality tests also revealed abnormalities in the amorphous head grouping of the sperm of male mice exposed to monoglyme at 500 ppm. Tonkin et al. (2009) us ed an Expression Analysis Systematic Explorer analysis of 28 genes in testicular samples
9 exposed to 2 methoxyethanol He showed statistical enrichment in genes that included protein transport, endocytosis, protein kinase activity, cell cycle, and meiosis. Also, increased expression of the actin binding protein cortactin and the transcription factor Finally, the Environmental Protection Agency (2001) reported that monoglyme treated pregnant female rats at 120 mg/kg/day were associated with 100% of fetal death. Doses of 30 or 60 mg/kg/day were associated with fetotoxi city but no major malformations were found. Diglyme. Similar to monoglyme, diglyme has favorable chemical properties that allow it to be used in a wide range of applications. Diglyme is miscible with water and organic solvents. It is chemical ly inert and has preferable solvent properties. D iglyme is used in applications that include Grignard, reduction, alkylation, and organometallic Neil, 2001; Environmental Protection Agency, 2003). Diglyme is also used in the coating industry and in photolithography in the manufacturing of semiconductor chips (Corn & Cohen, 1993; Correa et al., 1996) Diglyme can be synthesized from reacting ethylene oxide with methanol in the presence of either acidic of basic catalysts, as well as from diethylene glycol and dimethyl sulfate (Environmental Protection Agency, 2003). The structure of diglyme is shown in Figure 1 4. Diglyme differs from monoglyme by having an additional ethoxy group in the middle of the structure. Metabolism studies in rats and mice have shown that diglyme is rapidly absorbed from the gastrointestinal tract (Cheever et al., 1988; Cheever et al., 1989; Daniel et al. 1991). Inhalation studies showed that diglyme is absorbed from the lungs a nd resulte d in poisoning symptom s in studies on single and Figure 1 4 Diglyme (U.S. National Library of Medicine, 2012)
10 repeated dose exposures. Also, in vitro dermal absorption studies with human skin revealed that diglyme has one of the highest absorption rates among glycol ethers (Johanson, 1996; Filon et al., 1999). This hints that in vivo absorption should also be expected. Diglyme m etabolites have been well studied in rats. Diglyme can be metabolized through two different pathways. The main pathway occurs through O demethylation followed by oxidation through the correspo nding aldehyde which leads to the major metabolite 2 ( 2 methoxyethoxy ) acetic acid. This metabolite consisted of 70% of the dose in the urine of rats and pregnant mice (Toraason et al., 1996). After another oxidative demethylation to 2 hydroxyethoxyacetic acid, diglycolic acid is formed and excreted. This process, shown as pathway B in F igure 1 5, is catalyzed by an unspecified cytochrome P 450. Alternatively, diglyme can be cleaved at the central ether bond through O dealkylation to form 2 methoxyethanol. This is catalyzed by cytochrome P 450 II E1 as outlined in pathway A of F igure 1 5. After ward 2 methoxyethanol is oxidized to 2 methoxyacetic acid which makes up about 5 15% of dose in the urine rats (Cheever et al., 1988, 1989). Most of the 2 methoxyaceti c acid is excreted through urine but some is conjugated with glycine to produce N methoxyacetyl glycine. In pregnant mice, 2 methoxyacetic acid made up nearly 28% of the dose (Daniel 1991; World Health Organization, 2003). However, human microsomes were mo re efficient in converting diglyme to 2 methoxyethanol than rat micromes (Tirmenstein, 1993; Toraason et al., 1996). Cheever et al. (1988) concluded that there is no crossover from 2 (2 methoxyethoxy)acetic acid to form 2 methoxyacetic acid. The metabolic route is dependent of the initial chemical metabolite formed and dependent on the levels of various cy tochrome P 450 isozymes present.
11 Figure 1 5 Metabolism pathway of diglyme (Envir onmental Protection Agency, 2003). Figure 1 6 (A) Section of testis of a control rat sacrificed. (B) Section of testis of rat exposed to diglyme after 10 days. Note loss of germinal epithelial cells and presence of spermatid giant cells and necrotic cell debris in seminif erous tubules (Valentine et al 1999).
12 The major metabolite of diglyme is 2 methoxyethoxyacetic acid. S everal studies indicate that 2 methoxyacetic acid metabolite is responsible for the toxicity of diglyme to the male reproductive organs (Cheever et al 1988; BUA, 1993). The World Health Organization (2003) reported that mean body weights of rats exposed to diglyme decreased over a 14 day period. Testicular m icroscopic examination by Valentine et al. (1999) sho wed severe testicular atrophy at the highe st exposure group as seen in F igure 1 6. All stages of sperm maturation were affected and included spermatid formation and germimal cell exfoliation. Furthe rmore, rat haematopoietic system is also affected by diglyme. The number of platelets, monocytes an d neutrophils were reduced in exposed rats. This suggests that diglyme toxicity is also due to an interaction with bone marrow. However, both testicular and hemaetopoeitic effects were nearly reversible after 42 days. Rats exposed to 2 methoxyethanol showe d more severe testicular pathology than when exposed to equal concentrations of diglyme The main metabolite of 2 methoxyethanol is 2 methoxyacetic acid (Cheever et al., 1988) Unlike monoglyme, several rat studies reported that acute inhalation exposure to diglyme did not result in fatalities during exposure and up to 14 days after (McGregor et al., 1983; Valentine et al., 1999). Restlessness, narrowing of palpebral fissu res, and irregular breathing were observed. Repeated dose inhalat ion studies with rats revealed that males were more sensitive than females. In vitro genetic toxicology studies using Salmonella typhimurium reverse mutation assays were negative. Also, the metabolic activating systems of rat and hamster livers were not fo und to have any mutagenic activity (Mortelmans et al., 1986). However, a study by Driscoll et al. (1998) showed that diglyme and 2 methoxyethanol were developmental toxins. Pregnant rats were exposed
13 to 25 ppm 2 methoxyethanol and 25, 100, and 400 ppm digl yme for 6 hours/day through days 7 16 of gestation or on gestation day. The group exposed to 400 ppm diglyme showed decreased food consumption and no live fetuses. At 100 ppm diglyme, embryo viability was unaffected but fetal weight s were lower than contro l groups. M aternal rats showed increased liver weights in the 100 ppm group In comparison, 2 methoxyethanol induced decreased food consumption and increased liver weights at 25 ppm. All diglyme exposed groups showed fetus structural malformations M alform ations were characterized by delayed skeletal ossification and rudimentary ribs. While fetal malformation was detected as low as 25 ppm diglyme, no maternal symptoms could be detected at this concentration. III. GLYCOL ETHER S The National Institute of Environmental Health Sciences nominated ethylene glycol 2 ethylhexyl ether for toxicological characterization because of its similarity to other known toxic ethylene glycol ethers, its widespread use, and unknown toxicity profile (National Tox icology Program, 2008b) The National Toxicology Program states that only ethylene glycol 2 ethylhexyl ether acute LD 50 data are available. However, the National Toxicology Program has studied other glycol ethers like ethylene glycol monobutyl ether which has a similar structure to ethylene glycol 2 ethylhexyl ether (Environmental Protection Agency, 2010) Furthermore, studies on ethylene glycol monohexyl ether, which differs from ethylene glycol 2 ethyhexyl ether by only an ethyl group, have also been cond ucted (California Environmental Protection Agency, 2010) The metabolism of ethylene glycol monobutyl ether is well documented in rodents Hu man metabolism is also available. One of the main metabolites, 2 butoxyacetic acid,
14 was identified as a hemolytic t oxin as early as 1956 by incubation with whole blood from different species (Carpenter et al., 1956). The glycol ethers 2 methoxyethanol and 2 ethoxyethanol which are structurally smaller than ethylene glycol monobutyl ether, have been identified as testi cular and developmental toxins (National Toxicology Program, 1993). The possibility of structurally similar and larger glycol ethers than ethylene glycol monobutyl ether being metabolized into toxi c products remains largely unknown due to lack of studies The increase in production of ethylene glycol 2 ethylhexyl ether will lead to greater human exposure and the lack of t oxicology studies is concerning. T he decision to study ethylene glycol 2 ethylhexyl ether by the National Institute of Environmental Health Sciences is understandable and necessary to answer toxicity concerns (National Toxicology Program, 2008a) A. ETHYLENE GLYCOL MONOBUTYL ETHER Chemical and Physical Information. Ethylene glycol monobutyl e ther, also known as 2 butoxyethanol, is used in a wide variety of applications. It is most commonly used as an industrial solvent in surface coatings, cleaning products varnish removers, and latex paints ( Environmental Protection Agency, 2010; Hung et al. 2011; Raymond et al., 1998; Veulemans et al., 1987; Wieslander & Norback, 2010 ) Adverse health effects like asthma have been associated with cleaning sprays, furniture polish and oven sprays ( Bello et al., 2009; Zock et al., 2001). A study by Wu et al. (2011) to investigate indoor air quality of small and medium sized commercial buildings in California showed that 2 butoxyethanol was found in high concentrations in paints and adhesives. The
15 Environmental Protection Agency (2010) estimated that household products contained 2.6 % ethylene glycol monobutyl ether in 1977. Ethylene glycol monobutyl ether is considered a high production volume chemical with an estimated 390 million pounds produced in the United States in 1992 (National Toxicology Program, 2000) Ethylene glycol monobutyl ether is a colorless liquid at ambient temperature and pressure. It is described as having a mild, ether like sweet odor (Hazardous Substances Data Bank, 2012). One of the main properties that make 2 butoxyethanol a widespread use chemical is its solubility with other substances. It is miscible in most organic solvents such as ethyl alcohol, ethyl ether, mineral o il, acetone, benzene, and heptanes (Flick, et al., 1985). It is miscible in all proportions with many keto nes, alcohols halogenated carbons, and ethers. It is slightly soluble in carbon tetrachloride, but also miscible in water. The structure of ethylene glycol monobutyl ether, displayed in Figure 1 7, shows that it contains both hydrophilic and lipophilic groups making it an amphiphile. Its amphiphile property explains why it is miscible in such a wide range of chemicals. Ethylene glycol monobutyl ether is considered a slow evaporator with an evaporat ion rate relative to butyl acet ate being 0.08 (Environm ental Protection Agency, 2010). Physical and chemical properties of 2 butoxyethanol are included in T able 1 2. Figure 1 7 Ethylene Glycol Monobutyl Ether (Hazardous Substances Data Bank, 2012).
16 Table 1 2. Physical and Chemical Properties of Ethylene Glycol Monobutyl Ether (Environmental Protection Agency, 2010; Hazardous Substances Data Bank, 2012). CASRN 111 76 2 Molecular Weight 118.2 Molecular Formula C 6 H 14 O 2 Vapor Pressure 0.88 mm Hg at 25C (about 1,200ppm) Flash Point 63C (closed cup); 70C (open cup) Boiling Point 168.4C Solubility Soluble in all proportions with ketones, alcohols, halogenated carbons, and ethers; miscible in water Distribution. The distribution and excretion of ethylene glycol monobutyl ether has been studied in rats. Bartnik et al. (1987) used subcutaneous doses of [ 14 C] 2 butoxyethanol to examine radioactivity in rat excretes and tissues. Within 72 hours of exposure, nearly 80% of radioactivity was detected in the urine, 10% in expired air, and 0.5% in feces. Compared to the blood, the spleen and thymus showed elevated levels of radioactivity. Topical application of [ 14 C] 2 butoxyethanol to rats showed that about 28% of the dose was absorbed within 24 hours with 19% of the radioactivity detected in urine, 6% in expired carbon dioxide, 0.4% feces, and 1.3% in the carcass (Lockley et al., 2004) The majority of 2 However, only 0.5% unchanged 2 butoxyethanol was detected suggesting that significant metabolism of this compound is occurring. Metabolism and Excretion. There are two major pathways by which ethylene glycol monobutyl ether is metabolized in humans and rats. The metabolic processes of rats and humans share similarities and differences that are highlighted in their elimination of ethylene glycol monobutyl ether. One pathway involves the conversion of ethylene glycol
17 monobutyl ether into the transient intermediate 2 butoxyacetaldehyde by alcohol dehydrogenase followed by its conversion to 2 butoxyacetic acid by aldehyde dehydrogenase (Corley et al., 1997; Corley et al. 2005; Medinsky et al., 1990) Corley et al. (2005) reported that 2 butoxyacetic acid is found in rat and human urine aft er exposure to 2 butoxyethanol. Human c onjugate d forms of 2 butoxyacetic acid include glutamine and glycine amide bound to the carbon y l end of the molecule forming an amide bond T he conjugated glutamine metabolite ma kes up the majority of 2 butoxyacetic a cid in humans; Corley et al. (1997) reported that 67% of urinary 2 butoxyacetic acid collected from humans after one arm exposure to 2 butoxyethanol was conjugated with glutamine. A different study by Rettenmeier et al. (1993) that analyzed the urine of la cquerers exposed to 2 butoxyethanol revealed that 90% of the 2 butoxyacetic acid conjugate was also N butoxyacetylglutamine. The second pathway involves O dealkylation by cytochrome P450 2E1, a cytochrome P450 dealkylase (Corley et al., 1997; Medinsky et al., 1990). The metabolites of this second pathway include ethylene glycol as well as a glucuronide and sulfate conjugate of ethyle ne glycol monobutyl ether. However, these metabolites have only been detected in rat urine (Bartnik et al., 1987 ; Ghanaye m et al., 1987 ). Figure 1 8 shows the metabolic pathways of ethylene glycol monobutyl ether.
18 Figure 1 8 Metabolism Pathway of Ethylene Glycol Monobutyl Ether, EGBE (Environmental Protection Agency, 2010). Ghanayem et al. (1987) used pyrazole and cyanamide to study the metabolic pathways of ethylene glycol monobutyl ether. Pyrazole and cyanamide are metabolic inhibitors of alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Ghanayem et al. (1987) treated male rats with cyanamide and pyrazole followed by administration of ethylene glycol monobutyl ether. The pyrazole treated rats had a 10
19 fold lower ratio of 2 butoxyacetic acid to conjugated ethylene glycol monobutyl ether and were protected from hematotoxicity. The cyanamide treated rats also showed reduced h ematotoxicity but resulted in high er mortality rate s This was likely due to the high levels of unmetaboliz ed 2 butoxyethanal. Ghanayem et al. (1987) confirmed that 2 butoxyacetic acid p lays a major role in the observed rat and human hematotoxicity Hematotoxicty. The metabolite 2 butoxyacetic acid causes hemolysis, the rupture of red blood cells (Bartnik et al., 1987 ; Ghanayem et al., 1987 ; Corley et al., 1994). While the exact mecha nism of hemolysis by 2 buto xyacetic acid remains unknown, Udden and Patton (2005) provides important mechanistic clues. Rat erythrocytes were incubated with 2 butoxyacetic acid and sucrose to determine the effect of the osmolarity of the external environme nt. Control experiments included rat erythrocytes incubated with sucrose but without 2 butoxyacetic acid. T hese cells showed a slight decrease in cell volume and no hemolysis Cells incubated with 2.0 mM 2 butoxyacetic acid but without sucrose show ed incre ase cell volume and about 5% hemolysis. W hen cells were treated with 2 butoxyacetic acid and an increasing sucrose gradient the increase in mean cell volume was not observed and hemolysis was reduced. However, erythrocytes swelled again when removed from the sucrose medium and resuspended in isotonic medium. R esuspension of the cells in the isotonic medium increased hemolysis from 1.5% to 6.6% Thus, Udden and Patton (2005) concluded that sucrose pr ovides osmotic protection without directly inhibiting the action of 2 butoxyacetic acid Udden and Patto n (2005) also investigated in reducing h emolysis. When incubating rat erythrocytes with 2.0 mM 2 butoxyacetic acid and replacing external sodium with potassium in the absence of calcium, hemolysis was greatly
20 reduced In normal conditions where calcium is present, erythrocyte hemolysis incubated with 2.0 mM 2 buto xyacetic acid was about 5% in the presence of sodium. H owever, the removal of calcium corresponded to an increase of 70 80% hemolysis. Based on these results, Udden and Patton (2005) further tested the effect of calcium on hemolysis. Udden and Patton (2005) incubated rat erythrocytes with 2.0 mM 2 butoxyacetic acid in the presence of CaCl 2 and MgCl 2 divalent cations. Ca lcium reduce d hemolysis at a concentration as low as 0.05 mM while magnesium had no protective effect. When ethylene glycol tetraacetic acid, a calcium chelating chemical, was added to the media with CaCl 2 hemolysis increased in the presence of ethylene g lycol monobutyl ether. In the presence of calcium, there was a dose dependent effect of 2 butoxyacetic acid on hemolysis and cell volume. In the absence of calcium, hemolytic dose response s were greater but there was no dose response effect for increase d c ell volume. Udden and Patton (2005) also showed that calcium delay ed hemolysis. Red blood cells incubated with 2 butoxyacetic acid and calcium experienced significant delayed hemolysis compared to erythrocytes without calcium as shown in Figure 1 9 g ra p hs A and B Figure 1 9 graphs C and D, show that mean cell volume during the first hour of incubation was not dependent on calcium. After significant hemolysis occurs, the mean cell volume of the remaining cells appears to be smaller. Furthermore, atomic abso rption spectroscopy showed an increase in calcium remaining in the red blood cells after exposure to 2 butoxyacetic acid in the presence of calcium. These observations led Udden and Patton (2005) to hypothesize that calcium activat es the Gardos pathway instead of having a direct interaction with 2 butoxyacetic acid (Gardos, 1958) The Gardos pathway is a potassium channel that prevents cell swelling by releasing potassium
21 through calcium activation. Udden and Patton (2005) tested th is hypothesis by using charybdotoxin, a scorpion toxin that inhibits Figure 1 9 Effect of calcium o n hemolysis and mean cell volume, MCV. Hemolysis of rat erythrocytes incubated with 1.0 mM (panel A) or 2.0 mM (panel B) 2 butoxyacetic acid and buffer c ontaining 0 mM ( ) or 2.0 mM ( ) calcium is shown. Panel C shows mean cell volume changes in 1.0 mM 2 butoxyacetic acid while panel D shows incubations with 2.0 mM 2 butoxyacetic acid. calcium activated potassium channels (Wolff et al., 1988). Rat erythrocyte hemolysis was not affected by charybdotoxin incubation. However, nanomolar concentrations of charybdotoxin caused a dramatic increase in hemolysis when erythrocytes were incubated with 2.0 mM 2 butoxyacetic acid in calcium containing buffe r. Similar degrees of hemolysis wer e observed when red blood cells were incubated with charybdotoxin and calcium, and without calcium, in the presence of 2 butoxyacetic acid. These observations
22 suggest that external calcium delays hemolysis in the presence of 2 butoxyacetic acid by a mechanism through the Gardos channel Ethylene glycol monobutyl ether is the most studied of the three glycol ethers investigated in this thesis project Next, we take a look at ethylene glycol monohexyl ether which only diff ers from to 2 butoxyethanol by a hydrocarbon chain that is two carbons longer. B. ETHYLENE GLYCOL MONOHEXYL ETHER Chemical and Physical Information. Ethylene glycol monohexyl ether, also known as 2 hexoxyethanol, is a high boiling solvent used in similar applications as 2 butoxyethanol (California Environmental Protection Agency, 2003). These applications include surface coatings, cleaning solutions, latex paints, and specialty printing inks (DOW, 2007). It is used for its excellent oil solubil ity. Exposure to vapors at room temperature is unlikely because of its high boiling point and slow evaporation rate, (DOW, 2007). However, Zhu et al. (2001) detect ed 2 hexoxyethanol in the emissions of three sample consumer cleaning products. Exposure to e thylene glycol monohexyl ether is mostly limited to industrial workplace s; however, Zhu et al. (2001) show ed that consumer exposure is possible from cleaning products Ethylene glycol monohexyl ether is described as having a pungent odor (DOW, 2007). It is a clear liquid that has limited water solubility and excellent oil solubility. It is produced from the reaction of ethylene oxide with anhydrous n hexyl alcohol, as shown in Figure 1 10 (DOW, 2007). In 2002, the estimated volume of ethylene glycol monoh exyl ether produced in the United States was between 454 and 4,540 metric tons
23 (California Environmental Protection Agency, 2010). In 2004, the United States consumed 309,000 metric tons of all ethylene glycol ethers (DOW, 2007). Table 1 3 shows ph ysical and chemical properties of ethylene glycol monohexyl ether. Table 1 3 Physical and Chemical Prope rties of Ethylene Glycol Monohex yl Ether (DOW, 2007; Hazardous Substances Data Bank, 2012). CASRN 112 25 4 Molecular Weight 146.23 Molecular Formula C 8 H 18 O 2 Vapor Pressure 0.05 mm Hg at 25C Density 0.8894 at 20C Boiling Point 208 C Solubility Slightly soluble in water; very soluble in alcohols, ethers, and oils Toxicity. Reports of ethylene glycol monohexyl ether acute human exposure are limited to high level exposures due to extreme large ingestions (California Environmental Protection Agency, 2010). Effects caused by these ingestion s include central nervous system depression, renal injury, hyperventilation, hemolysis, and metabolic acidosis. Boatman and Knaak (2001) report ed that 2 hexoxyethanol causes severe injury to the eyes and skin irritation while DOW (2007) reports that extend ed skin contact can result in absorption of harmful doses Figure 1 10 Synthesis of ethylene glycol monohexyl ether. (DOW, 2007).
24 Most toxicological information on ethylene glycol monohexyl ether is found on animal acute exposure studies. Smyth et al. (1954) reported rat dermal LD 50 as 0.89 m L /kg body weight. Ballantyne and Meyers (1987) reported LD 50 of 2 hexoxyethanol for male and female rabbits as 0.81 mL/kg and 0.93 mL/kg, respectively. Studies on male and female New Zealand White rabbits and male and female rats by direct application on the skin and intra venous route have also been performed using [ 14 C] 2 hexoxyethanol (Ballantyne et al., 2003). Intravenous application resulted in LD 50 values of 53.6 mg/kg and 70.0 mg/kg for male and female rats, respectively. Male and female rabbit LD 50 through intravenou s exposure were lower than rats. Male rabbit LD 50 was 40.0 mg/kg while female rabbit LD 50 was 30.3 mg/kg. Ballantyne et al. (2003) reported that death times for rats and rabbits were rapid with most being under 10 minutes. Application on the skin caused ir ritation and edema as well as increased body weight in animals that survived the lower exposure dose s. Male rat o ral exposure LD 50 were reported by Smyth et al. (1954) as 1.48 g/kg and 1.49 g/kg by Ballantyne and Myers (1987). Female rats displayed a l ower oral LD 50 of 0.74 g/kg (Ballantyne and Myers, 1987). Rats administered ethylene glycol monohexyl ether by oral route displayed sluggishness and unsteady walking (UNEP, 2003). Smyth et al. (1954) and Ballantyne and Myers (1987) reported no signs of 2 hexoxyethanol toxicity or lethality after inhalation exposure to saturat ed air for up to 8 hours. Absorption, Distribution, Metabolism and Excretion Ballantyne et al. (2003) provide the most current and complete metabolism studies on ethy lene glycol monohexyl ether found in the literature. Intravenous application of [ 14 C] 2 hexox yethanol in rats revealed that most radioactivity was found in the excreta. Initial urine analysis using high pressure
25 liquid c hromatography did not detect free et hylene glycol monohexyl ether However, 96% of radioactivity was detected in the void volume of the column meaning that metabolites were present but the column was not ideal for retention Urine high performance liquid chromatography using an anion exchan ge column revealed 2 7 peaks that did not correspond to the retention time of ethylene glycol monohexyl ether. Ballantyne et al. (2003) used glucuronidase to free any conjugates but reanalysis still did not reveal an ethylene glycol monohexyl ether peak. Plasma concentrations of ethylene glycol mono hexyl ether were not detectable after 8 hours. I ntravenous administration of [ 14 C] 2 hexoxyethanol in rabbits as in rabbits, revealed that radioactivity recovery was mostly found in urine, 78 83% over 48 hours but most being recovered in the first 24 hours (Ballantyne et al., 2003). Analysis with the anion exchange column detected 3 4 major peaks and 1 4 scattered minor peaks. Plasma concentrations of ethylene glycol monohexyl ether were not detectable at 1 hour post dosing and up to 48 hours thereafter (Ballantyne et al., 2003). Interestingly, percutaneous administration of ethylene glycol monohexyl ether resulted in differen t excretion routes than with intravenous administration (Ballantyne et al., 2003). Skin application of [ 14 C] 2 hexoxyethanol in rats resulted in a higher proportion of radioactivity excretion through feces Urine was the major excretion route with about 33 % and 21% in male and females rats, respectively. Gender differences were observed in radioactive plasma values with 17 55.37% detected in female rats and 5.78 36.29% in males. These wide ranges overlapped but showed that female rats had a larger percent o f radioactivity in plasma. The anion exchange column detected three major
26 peaks and four minor peaks, with no gender differences, but the peaks could not be identified (Ballantyne et al., 2003). Percutaneous exposure i n rabbits was different from rats. T he major radioactivity recovery was 58 68% in urine. The majority of elimination occurred w ithin the first 24 hours. H igh performance liquid chromatography of urine detect ed up to 3% unchanged ethylene glycol monohexyl ether in one female rabbit. Most of t he radioactivity was detected in the void volume. The anion exchange column detected three major peaks and up to six minor peaks (Ballantyne et al., 2003). Free ethylene glycol monohexyl ether plasma levels were transient and remained low with no detection 1 hour after dosing. In summary, ethylene glycol monohexyl ether studies are mainly limited to animal toxicity studies and few metabolism studies. Toxicity LD 50 studies show ed that there are gender differences that require further investigation (Ballantyne and Myers, 1987). Excretion routes were affected by method of exposure with urine being the most common route of excretion, though proportions varied based on method of expos ure. Also, ethylene glycol monohexyl ether metabolite peaks were detected but could not be identified (Ballantyne et al., 2003). C. ETHYLENE GLYCOL 2 ETHYLHEXYL ETHER Chemical and Physical Information. The National Institute of Environmental Health Sciences nominated ethylene glycol 2 ethylhexyl ether for toxicological studies due to its unknown toxicity and structural similarity to other know n toxic ethylene glycol ethers (National Toxicology Program, 2008b) In addition, the production volume in the United States increased from 10,000 to 500,000 pounds in 1990 to 1 to 10 million pounds in
27 2002 (National Toxicology Program, 2008a). A study by T akeuchi et al. (2005) reported that ethylene glycol 2 ethylh exyl e ther w as one of eleven indoor air chemicals in the house of a patient with multiple chemical sensitivity The chemical structure of ethylene glyco l 2 ethylhexyl ether is shown in F igure 1 11 Ethylene glycol 2 ethylhexyl ether is a n amphiphilic, clear liquid, high boiling solvent used in a wide variety of applications (National Toxicology Program, 2008a) It is found in acrylic glossy pain ts, ink jet inks, sunburn treatment and oral hygiene products, lens manufacturing cleaning solution, and as an antimicrobial in paper products and shampoos, among other uses (Diamond Vogel Paint, 2004; Choy, 2003 pat. appl.; Greff, 1998 pat. appl.; Nishiha ra and Wada, 2001 pat. appl.; Lynch et al., 2005 pat. appl.; Saud et al., 2004 pat. appl.). The most common route of synthesis is from the reaction of 2 ethylhexanol and ethylene oxide in the presence of mont morillonite or hydrotalcite as catalyst s (Fujita et al., 1987 pat. appl.; Maruyama et al., 2002 pat. appl.) Table 1 4 displays chemical and physical properties of ethylene glycol 2 ethylhexyl ether Table 1 4 Physical a nd Chemical Prope rties of Ethylene Glycol 2 Ethylhexyl Ether (National Toxicology Program, 2008a). CASRN 1559 35 9 Molecular Weight 1 74.28 Molecular Formula C 10 H 22 O 2 Vapor Pressure 0.0179 mm Hg at 25C Density 0.88 at 20C Boiling Point 229 C Solubility Slightly soluble in water Figure 1 11 Chemical structure of ethylene glycol 2 E thylhexyl ether (National Toxicology Program, 2008a).
28 Toxicity T oxicological studies of ethylene glycol 2 ethylhexyl ether are limited to acute exposure and few subchronic exposure investigations. Chronic exposure studies could not be found in the literature. The Nati onal Toxicology Program (2008a) reports that acute exposure to ethylene glycol 2 ethylhexyl ether resulted in an oral LD 50 of 3898 mg/kg for mice and 4600 mg/kg for rats Effects on mice included general depressed activity, tremors, and labored breathing. In rabbits, the dermal LD 50 was 2584 mg/kg and effects of exposure included anorexia, depression, and soft fec es at low doses. A t higher doses, nasal discharge, labored breathing, and complete physical exhaustion w as observed. The National Toxicology Progr am (2008a) reports that rats exposed subchronic all showed an oral dose LD 50 of 117 g/kg ethylene glycol 2 ethylhexyl ether After six weeks rats showed general depressed activity, pigmented or nucleated red blood cells, and changes in erythrocyte count (National Toxicology Program, 2008a). Reproductive and developmental toxicity studies using a series of glycol ethers with a dose range of 1.5 160 k/kg for six weeks found that ethylene glycol 2 ethylhexyl ether resulted in testicular atrophy and degenerative spermatozoa (National Toxicology P rogram, 2008a). This same study assessed hematotoxicity and found that ethylene glycol 2 ethylhexyl ether induced reduced red blood cells and reduced hemoglobin. Mechanistic information for these effects is not known. Genetic toxicity, neurotoxicity, carci nogenicity, and immunotoxicity data are not available for ethylene glycol 2 ethylhexyl ether (National Toxicology Program, 2008a). Metabolism. The re are no metabolism ethylene glycol 2 ethylhexyl ether studies found in the literature. The la ck of ethylene glycol 2 ethylhexyl ether metabolism information is the main basis for this project. The liver is the main site of metabolism of foreign
29 compounds (Caldwell et al., 1995). Cryopreserved hepatocytes are a good tool for the prediction of metabolic clearance since they retain enzymes responsible for phase I and phase II biotransformations (McGinnity et al., 2004). Phase I reactions involve functionalization reactions which result in making a functional group suitable for further metabolism an d excretion. Common phase I reactions are performed by alcohol and aldehyde dehydrogenase. Phase 2 reactions occur when conjugation with an endogenous conjugating agent occurs. Common Phase 2 reactions include conjugation with glucuronic acid, sulfate, methylation, and acetylation. In this experiment, we were mainly concerned with determining the half life of each glycol ether using cryopreserved hepatocytes from humans, mice, and rats of both sexes to assess differences between species and gender. These results will be examined to determine which animal model will reproduce metabolic data most similar to humans. Also, method development for detecting phase I and phase II metabolites from this in vitro model will be examined.
30 CHAPTER 2 MATERIALS & METHODS I. H IGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY LC/MS/MS system will help clarify the data collected for this project. An Applied ystem wa s used to detect, separate, identify and quantify glycol ethers during method development and after incubation with cryopreserved hepatocytes at different time points. The API 5000 is a triple quadrupole that separates ions according to their mass to charg e ratios by imposing DC and RF voltages. The first tri ple quadru pole, termed Q1, separates ions before they enter Q2. Q2 is a collision cell in which ions can be broken into fragments by collisions with gas molecules. The fragments from Q2 enter Q3 for add itional separation The ions from Q3 enter the detector and create a current that is converted into a voltage pulse The instrument converts voltage pulses which are directly proportional to the quantit y of ions entering the detector and converts the infor mation into a signal. This filtering mode, termed Multiple Reaction Monitoring, is shown in Figure 2 1.
31 Figure 2 1. MS/MS Multiple Reaction Monitoring. Q1 is a precursor ion filter; Q2 is a collision cell where fragments of the precursor ion are made by collisionally activated dissociation (CAD) gas; Q3 filters fragment ions that proceed to the detector (University of Guelph). In this experiment, Atmos pheric Pressure Chemical Ionization (APCI ) was used to generate the precursor ions The components of an APCI source are shown in Figure 2 2 (Paul Gates, 2004). The analyte originating from the liquid chromatography column is introduced into a reaction ch amber through a heated nebulizer which causes the solvent or mobile phase to evaporate. T he mobile phase solvents in this experiment were water and methanol. A corona discharge needle caused electrons to be removed from the nebulizer gas, into a hydrocarbo n free atmosphere zero air gas, resulting in the primary N 2 + and O 2 + ions (Paul Gates, 2004; University of Guelph). A complex set of reactions shown below, produces protonated solvent (Paul Gates, 2004).
32 Figure 2 2 Schematic of the components of an APCI source (Paul Gates, 2004). The mobile solvents, water and methanol, resulted in corona discharge H 3 O + and CH 3 OH 2 + reagent ions The formation of these protonated reagent ions is further assisted by adding 0.1% formic acid by volume to each of the mobile phase solvents. The protonated analyte ions are formed by gas phase acid base ion molecule reactions and dependent on the gas phase basicity and acidity of the species involved (Polettini, 2006). Figure 2 3 depicts the corona discharge reaction and collision between reagent ions and analyte molecules. Glycol ethers gain a positive charge by protonation with the solve nt reagent ions forming [M+H] + which effectively increases the molecular weight of the glycol ethers by 1 atomic mass unit. For exam ple, the molecular weight of ethylene glycol monobutyl ether is 118.17 so the protonated form would have a molecular weight of 119.17 and this would be the input value for Q1 when filtering for this protonated ion. When scanning for positively charged mole cular ions, the mass spectrometer is running in positive mode.
33 A. PARAMETERS Several parameters can be adjusted to optimize detection of the analyte. A list of these parameters and their function can be found in Appendix A This information comes al which can be found online (see Applied Biosystems, 2005, in references). The effect of changing parameters can best be seen by looking at the RO2 (Collision Cell Rod Offset) parameter which is better known as the Collision Energy. This parameter contr ols the potential applied to the collision cell, Q2. The Collision Energy is the potential difference between Q0 and Q2. This is the amount of energy that the precursor ions receive as they are accelerated into the Q2 collision cell, where they collide wit h gas molecules and fragment. Refer to Figure A 1 of the appendix to see where Q0 and Q2 are in relation to each other. A Collision Energy examination can be Figure 2 3. Mechanism of APCI. Corona discharge needle produces solvent reagent ions that collide with analyte molecules, M, and transfer a positive charge by protonation (Gates, 2004).
34 performed while chemicals are introduced into the mass spectrometer by a syringe pump. For exampl e, a Collision Energy examination of ethylene glycol 2 ethylhexyl ether is shown in Figure 2 4. A Collision Energy scan from 5 to 41 Volts was performed, as can be seen from the x axis of Figure 2 4A; the y axis shows the intensity, in counts per second, of all ions detected. Figure 2 4 B shows the ions detected when the Collision was 8 V. Here, we can see the parent molecular ion detected at a m/z of 175.1 Da, which is the molecular weight of protonated ethylene glycol 2 ethylhexyl ether. The other m/z pe aks are the fragments of ethylene glycol 2 ethylhexyl ether when the Collision Energy is 8 V. Figure 2 4C shows the m/z peaks when the Collision Energy is 20 V; we can see that the intensity of the parent ion has greatly decreased and that of the fragment ions has increased as the Collision Energy is increased. Finally, a Collision Energy of 40 V complete ly f ragments the parent ion and some of the bigger fragment ed ions producing smaller fragment ed ions of 45 Da or lower. A Collision Energy examination is very useful because it show s fragment ions from a parent compound, and the optimal Collision Energy value to detect fragments Ethylene glycol monobutyl ether and ethylene glycol monohexyl ether fragmented ions are displayed in Figure A 2 of the appendix.
35 Figure 2 4. Effect of Collision Energy on protonated ethylene glycol 2 ethylhexyl ether parent and fragment ions. Graph A shows the total number of ions detected at different Collision Energies, measured in the x axis in Volts. Spectrum B shows the ions when the Collision Energy is at 8 V. Notice that the parent ion is seen with a m/z of 175.1 Da, which corresponds to protonated ethylene glycol 2 ethylhexyl ether. Spectrum C shows the result when the Collision Energy is increased to 20 V; the parent ion has greatly decreased and the fragment ions have increased. Spectrum C shows the ions, mainly fragment ions, when the Collision Energy is 40 V. Notice that not only has the parent ion disappeared but most fragments are small with a m/z of less than 45 Da.
36 II. EXPERIMENTAL PROCEDURES Materials. Ethylene glycol monobutyl ether ( ethylene glycol monohexyl ether ethylene glycol 2 ethylhexyl ether (97% purity) and the positive control 7 ethoxycoumarin ( 99.5% purity) were obtained from Sigma Aldrich (St. Louis, MO). The mobile phases OmniSol LC MS water and OmniSolv LC MS methanol were obtained from EMD Chemicals (Gibbstown, NJ). Formic acid (99% purity) was obtained from Thermo Fisher Scientific (Walth am, MA). Female and male human cryop reserved hepatocytes were obtained from CellzDirect (Carlsbad, CA). Female and male B6C3F1 mouse and Sprague Dawley rat cryopreserved hepatocytes were obtained from ALIVE (Albuquerque, NM). Williams Medium E, Hepatocyte Ma intenance Supplement Pack (Serum free) and Cryopreserved Hepatocyte Recovery Medium were obtained from Invitrogen (Carlsbad, CA). Hepatocyte Incubations and Sample Collection Williams Medium E was supplemented with Dexameth a sone in DMSO an d Cell Maintenance Cocktail from the Hepatocyte Maintenance Supplement Pack Williams Medium E was wa rmed to 37 C in a water bath. Ten microliters of 1 mM test article solutions (ethylene glycol monobutyl ether, et hylene glycol monohexyl ether, and ethylene glycol 2 ethylhexyl ether) in methanol was added to 4.99 ml of Williams Medium E to make 2 M test article solutions. A 2 M positive control solution of 7 ethoxycoumarin was made by adding 20 l of 1 mM 7 ethoxy coumarin stock solution in methanol to 10 ml of Williams Medium E. Forty eight m illi l iters of Cryopreserved Hepatocyte Recovery Medium was added to 50 ml conical tubes and warmed to 37 C in a water bath. Cryopreserved hepatocyte vials were taken from liqu id nitrogen storage and
37 immediately thawed in a 37 C water bath for 2 minutes. Vials were sprayed and wiped with 70% ethanol inside a hood and poured into warmed Cryopreserved Hepatocyte Recovery Medium. The Cryopreserved Hepatocyte Recovery Medium was ce ntrifuged (100 g for 10 minutes for human hepatocytes; 55 g for 3 minutes for mouse and rat hepatocytes) at room temperature. S upernatant was poured off. Twelve milliliters of warm Williams Medium E was added to the remaining hepatocyte pellet and gent ly swirled to suspend cells into solution. Fifty microliters of hepatocyte suspension was transferred to 50 l of diluted trypan solution (prepared by mixing 200 l of phosphate buffered saline with 0.4% trypan blue solution) in 2 ml microcentrifuge tube f or counting live cells Tube was gently tapped to ensure homogeneous mixture and cells were manually counted using a hemocytometer. Supplemented Williams Medium E was added to hepatoc yte solution to attain desired live cell yield s of 2.0 10 6 cells/ml. A heat deactivated negative control was made by boiling hepatocyte suspension for 5 minutes. A 12 well non coated plate containing the test articles was removed from the incubator and 500 l of the suspended hepatocyte Williams Medium E solution was added to respective wells. Five hundred microliters of 2 M test articles and positive control in Williams Medium E was added to respective wells of a 12 well non coated plate and placed on an orbital shaker, set to 100 rpm, inside an incubator at 37C and 5% CO 2 humidified atmosphere. A well of 500 l of Williams Medium E was also used as a no compound negative control. This produced wells with a total substrate concentration of 1 M and 1.0 10 6 viable cells per/ml. T he following wells were made for each hepat ocyte incubation, :
38 Duplicate wells of ethylene glycol monobutyl ether Duplicate wells of ethylene glycol monohexyl ether Duplicate wells of ethylene glycol 2 ethylhexyl ether Duplicate wells of 7 ethoxycoumarin positive control Single well of heat deactiv ated hepatocytes negative control Single well of no compound negative control At incubation time points of 5, 15, 30, 60, and 120 minutes hepatocytes were added to wells. Fifty microliter aliquots of well mixture was removed and added to microcentrifuge tubes containing 50 l of cold stop solution (0.1 M DMB internal standard and 0.1% formic acid in acetonitrile) and inverted multiple times B etween time points, plates were placed on orbital shaker inside incubator After all sample collec tion s w ere comp lete, microcentrifug e tubes were spun at 13,000 rpm for 5 minutes. Supernatants were collected into HPLC vials and stored at 8 0 C until analyzed. Instruments. High performance liquid chromatography analyses were performed on an Agilent (Santa Clara, CA) 1200 high performance liquid chromatograph. A Luna C18(2) column (3 m, 2.0 50 mm; Phenomenex, Torrance, CA) was used for ethylene glycol monobutyl ether meta bolism clearance studies. The mobile phase included 0.1% formic acid by volume in water (solvent A) and 0.1% formic acid by volume in methanol (solvent B). The elution began with a linear gradient from 10% B to 100% B over 4.00 minutes, then a 100% B isocr atic gradient over 3.00 minutes, followed by a rapid linear gradient back to 10% B over 0.10 minutes, and a 100% B isocratic gradient for 3.90 minutes for a total time of 11.00 minutes. The rapid linear gradient back to 10% B and the s ubsequent isocratic g radient were performed to equilibrate the column for the next
39 sample run. A 40.00 l injection from the HPLC vials was used with a flow rate of 400 l/min. The retention time of ethylene glycol monobutyl ether was 4.95 minutes. The retention time of the in dihydroxy 4 methoxybenzophenone was 6.28 minutes. A Synergi C12 with trimethylsilyl (TMS) endcapping column (2.5 m, 2.0 30 mm; Phenomenex, Torrance, CA) was used for ethylene glycol monohexyl ether and ethylene glycol 2 ethylhexyl ethe r metabolism clearance studies. T he no compound control, the heat deactivated negative controls, and the 7 ethoxycoumarin positive control were analyzed on the Synergi C12 column The same mobile phases wer e used as described above. E lution for ethylen e glycol monohexyl ether and ethylene glycol 2 ethylhexyl ether began with a linear gradient from 5% B to 100% B over 3.50 minutes. An isocratic gradient of 100% B was maintained for 1.20 minutes, followed by a rapid linear gradient back to 5% B over 0.10 minutes. A final isocratic gradient of 5% B was maintained for 0.90 minutes, for a total run time of 5.70 minutes, to equilibrate the column for the next sample run. A 40.00 l HPLC injection was used with a flow rate of 1200 l/min. The retention time of ethylene glycol monohexyl ether was 2.71 minutes, while that of ethylene glycol 2 ethylhexyl ether was 3.26 minutes. The retention time of the internal standard was 2.97 minutes. The Synergi C12 with TMS end capping column was also used to elute the positi ve control 7 ethoxycoumarin. Elutions began with a linear gradient from 5% B to 100% B over 0.50 minutes. An isocratic gradient of 100% B was maintained for 1.10 minutes, followed by a rapid linear back to 5% B. An isocratic gradient of 5% B was maintaine d for 1.00 minute to equilibrate the column for the next sample run for a total
40 time of 2.70 minutes. A 40.00 l injection from the HPLC vials was used with a flow rate of 1200 l/min. The retention time for 7 ethoxycoumarin was 1.40 minutes, and the reten tion time of the internal standard was 1.47 minutes. The temperature of the column compartment was maintained at 40.00C for all runs. APCI MS/MS was obtained on an API 5000 Triple Quadrupole LC/MS/MS Mass Spectrometer (Applied Biosystems, Foster City, CA ). Samples were introduced to the mass spectrometer directly after eluting from the HPLC column with 40.00 l injection volumes and the flow rates mentioned above for positive ionization analysis. Sample Analysis. Positive ion analysis of glycol ethers, po sitive control, and internal standard was performed using the API 5000 Triple Quadrupole LC/MS/MS in Multiple Reaction Monitoring mode. The source and compound dependent parameters used for each acquisition method can be found in Table A 1 of Appendix A. The following table displays the mass (in Daltons) of the parent ion compound filtered in Q1 and the fragment ions filtered through Q3 after collision of the parent compound with gas molecules in Q2. The fragment that had the highest intensity was chosen t o represent the amount of compound present. Some of the compound is lost since it can fragment into other species. However, fragments are proportional to the amount of compound entering Q1 enabling accurate measure s of remaining chemical different time poi nts.
41 Table 2 1. Mass of positive ions filtered through Q1 and Q3 chambers. Data Analysis. The software Analyst 1.5 (Applied Biosystems, Foster City, CA) was used to analyze the MS data. The glycol ethers response peak s were integrated to get an area under the curve and normalized according to the DMB internal standard MS response as shown in Figure 2 5 A glycol ether area : DMB internal standard area ratio is produced. The 5 minute time point was set to 100% and a percent remaining of the gly col ether was calculated for subsequent time points. The natural log of the percent remaining was used to calculate the half life of the test articles. Chemical Positive Ions Filtered Q1 Q3 Mass (Da) Ethylene glycol 2 ethylhexyl ether 175.0 63.0 Ethylene glycol monohexyl ether 147.1 63. 0 Ethylene glycol monobutyl ether 119.1 62.9 7 ethoxycoumarin 191.1 107.1 191.1 163.1 2,2' dihydroxy 4 methoxybenzophenone 245.0 121.1 245.0 151.1
42 Figure 2 5. Example integ ration of peaks using Analyst 1.5 software. The area under the DMB internal standard MS response peak is integrated (top spectrum), as well as the area under the glycol ether response peak (bottom spec trum). A ratio of these areas were calculated. The rati o for the 5 minute time point is set as the 100% remaining glycol ether.
43 CHAPTER 3 RESULTS I. HALF LIFE QUANTITATION The following spectra Figure 3 1, show s the detection peak for one of the trials of ethylene glycol 2 ethylhexyl ether using female human hepatocytes. Figure 3 1. Trial 2 spectra of ethylene glycol 2 ethylhexyl ether incubated with female human hepatocytes. Ethylene glycol 2 ethylhexyl ether detection peak is observed with a retention time of about 3.27 minutes (right hand peak in the samples from 15 minutes and longer) 120 minutes 60 minutes 3 0 minutes 15 minutes 5 minutes
44 Figure 3 1 shows that the detection peak with a retention time of about 3.27 minutes for ethylene glycol 2 ethylhexyl ether decreases as the incubation time increases. A detection peak with a retention time of 3.09 minutes is observed to increase as the incubation time increases. This was peculiar at first glance because the Q1 and Q3 filt ers were set to detect ethylene glycol 2 ethylhexyl ether, yet a signal appeared with a different retention time that shared the same Q1 and Q3 mass as ethylene glycol 2 ethylhexyl ether. In a later section, we investigate this further and discover it is a metabolite. Table 3 1 shows the peak ratios for both trials of ethylene glycol 2 ethylhexyl ether when incubated with female human hepatocytes. The peak ratios were calculated from the area under the peak of the 175.000/63.000 Q1/Q3 peak of ethylene glyc ol 2 ethylhexyl ether and the 245.000/151.100 peak of the internal standard 2,2' d ihydroxy 4 methoxybenzophenone Also displayed in Table 3 1 are the percent remaining and the natural log of the percent remaining of the time points and accompanied by graph s in Figure 3 2 and 3 3 as well as the k value which is the positive slope of the linear regression line, the calculated half life in minutes for each trial, and an average half life Figure 3 3 shows the linear regression lines for both trials. Table 3 1. Results from the incubation of female human hepatocytes with ethylene glycol 2 ethylhexyl ether
45 Figure 3 2. Percent remaining of ethylene glycol 2 ethylhexyl ether (EGEHE) incubated with female human hepatocytes. Each point represents one measurement. Figure 3 3. Natural logarithm of the percent remaining of ethylene glycol 2 ethylhexyl ether incubated with female human hepatocytes. The slope of the linear regression line was used to calculate the half life. 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 110 120 % Remaining Time (min.) Female Human EGEHE Clearance, % Remaining Trial 1 Trial 2 y = 0.0954x + 5.2961 R = 0.9371 y = 0.0727x + 5.0231 R = 0.9926 0 1 2 3 4 5 0 5 10 15 20 25 30 LN % Remaining Time (min.) Female Human EGEHE Clearance, LN % Remaining Trial 1 Trial 2
46 Table 3 2 shows the half l ives of all incubations with ethylene glycol 2 ethylhexyl ether (EGEHE), ethylene glycol monohexyl ether (EGHE), and the positive control 7 ethoxycoumarin (7 EC). Table 3 2. Half lives (in minutes) of all incubations with ethyle ne glycol 2 ethylhexyl ether (EGEHE), ethylene glycol monohexyl ether (EGHE), and the positive control 7 ethoxycoumarin (7 EC). Hepatocytes Trial t 1/2 (min) Average t 1/2 Standard Error E Female Human 1 7.27 8.40 1.13 2 9.53 G Male Human 1 3.80 5.26 1.46 2 6.71 E Female Mouse 1 4.65 5.76 1.11 2 6.87 H Male Mouse 1 6.51 7.75 1.24 2 8.98 E Female Rat 1 6.51 8.13 1.62 2 9.75 Male Rat 1 6.34 8.25 1.91 2 10.16 Female Human 1 44.67 144.13 99.47 2 243.60 E Male Human 1 45.51 84.14 38.63 2 122.78 G Female Mouse 1 60.96 70.69 9.73 2 80.42 H Male Mouse 1 68.42 63.92 4.50 2 59.42 E Female Rat 1 18.16 99.12 80.96 2 180.09 Male Rat 1 80.84 89.35 8.51 2 97.87
47 Table 3 2 (continued). Half lives of all incubations with ethylene glycol 2 ethylhexyl ether (EGEHE), ethylene glycol monohexyl ether (EGHE), and the positive control 7 ethoxycoumarin (7 EC). Hepatocytes Trial t 1/2 (min) Average t 1/2 Standard Error Female Human 1 9.43 9.47 0.03 2 9.50 7 Male Human 1 5.75 5.75 0.01 2 5.76 Female Mouse 1 3.73 3.67 0.07 E 2 3.60 Male Mouse 1 3.77 3.83 0.06 2 3.89 C Female Rat 1 83.48 93.21 9.74 2 102.95 Male Rat 1 146.50 160.31 13.81 2 174.11 The bar graphs in Figures 3 4, 3 5, and 3 6 show a comparison of the half lives. Figure 3 4 shows that the half life of ethylene glycol 2 ethylhexyl ether was nearly the same across all species and gender and fell within the standard error of each other. A significant difference was observed between female and male human hepatocytes. A n average 8.40 1.13 minutes female human hepatocytes half life did not fall within the standard error of the 5.26 1.46 minutes half life of male human hepatocytes. The 5.76 1.11 minutes half life of female mouse hepatocytes also did not fall within the standard error of the half life of female human hepatocytes. However, a one way ANOVA between all hepatocyte incubations reveals an F ratio of 0.91 meaning that there is no significant statistical difference among all the half lives
48 Figure 3 4. Half lives of all incubations with ethylene glycol 2 ethylhexyl ether (EGEHE). Half life variability was a bigger problem in the incubations with ethylene glycol monohexyl ether as Figure 3 5 shows. The standard error of the half lives of t he incubations with female human, male human, and female rat hepatocytes shows a wide range of variability between the two trials. In fact, all incubation half lives fall within the standard errors of these half lives. A clear difference was observed betwe en the ha lf life of female and male mice hepatocytes from the half life of male rat which again highlights a difference between species of the same gender. However, i t is clear that the large standard errors from these trials are a problem and need to be s maller for proper comparisons. A one way ANOVA was also performed for the half lives calculated for ethylene glycol monohexyl ether The calculated 0.27 F ratio suggests that there is no statistically significant difference between the half lives of all incubations. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 EGEHE Half life (min) EGEHE Average Half life Female Human Male Human Female Mouse Male Mouse Female Rat Male Rat
49 Figure 3 5. Half lives of all incubations with ethylene glycol monohexyl ether (EGHE). The calculated half lives of the positive control 7 ethoxycoumarin are represented graphically in Figures 3 6 and 3 7. The half lives for 7 etho xycoumarin show female and male human and mouse are below 10 minutes with a very small standard error s The average half life for female human hepatocytes was greater than that of male human hepatocytes when incubated with 7 ethoxycoumarin. The half lives for female and male mice were very similar Standard error bars did not overlap. F emale and male rat half lives with the positive control are shown graphically in Figure 3 7. These half lives are much greater than the human and mouse half lives. The female rat hepatocyte half life wa s less than the male rat and standard error bars did not overlap. The positive control clearly shows differences in metabolic clearance between species and gender. Interestingly, average half life of 7 ethoxycoumarin was higher in female human hepatocytes than male human hepatocytes. However, female rat half life 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 260.00 EGHE Half life (min) EGHE Average Half life Female Human Male Human Female Mouse Male Mouse Female Rat Male Rat
50 Figure 3 6. Half lives of female and male human and mouse hepatocytes with 7 ethoxycoumarin (7 EC). Figure 3 7. Half lives of all incubations with the positive control 7 ethoxycoumarin (7 EC). 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 Half life (min) 7 EC 7 EC Average Half life Female Human Male Human Female Mouse Male Mouse 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 1 Half life (min) 7 EC 7 EC Average Half life Female Human Male Human Female Mouse Male Mouse Female Rat Male Rat
51 was lower than in male rat hepatocytes, and there was only a small difference between female and male mouse hepatocytes. The ethylene glycol monobutyl ether incubation hal f lives could not be determined due to unexpected results. The percent remaining for most of the time points after 15 minutes showed an increase in the percent remaining from the 15 minute and even 5 minute time point. This was observed throughout all incu bations. Table 3 3 and Figure 3 8 show the percents remaining from the incubation of female human hepatocytes with ethylene glycol monobutyl ether. There are two possible explanations for these unexpected results. First, it is possible that an unidentified metabolite with the same retention time and fragmentation pattern is interfering with the detection peak of ethylene glycol monobutyl ether, as wa s observed with ethylene glycol 2 ethylhexyl ether and ethylene glycol monohexyl ether which will be Table 3 3. Data from the female human hepatocyte incubation with ethylene glycol monobutyl ether. The peak ratios increase after the 15 minute time point for trial 1 and after the 30 minute time point in trial 2. Time Point (minutes) Peak Ratio % Remaining LN % Remaining Trial 1 5 0.0704 100.00 4.61 15 0.0047 6.68 1.90 30 0.113 160.51 5.08 60 0.0931 132.24 4.88 120 0.127 180.40 5.20 Trial 2 5 0.166 100.00 4.61 15 0.152 91.57 4.52 30 0.11 66.27 4.19 60 0.167 100.60 4.61 120 0.16 96.39 4.57
52 Figure 3 8. Percent remaining of ethylene glycol monobutyl ether incubated with female human hepatocytes. The percent remaining increases after the 15 minute time point of trial 1 and the 30 minute time point of trial 2. discussed later. Second, the no compound negative control showed a detection peak with the same retention and fragments as ethylene glycol monobutyl ether as Figure 3 9 shows. An endogenous compound in the hepatocytes would be the likely cause of this. Contamination should not be ruled out but it is unlikely since the no compound wells were kept in separate plates from those with the test article. Figure 3 9. Retention peak of e thylene glycol monobutyl ether in the no compound negative control of female human hepatocytes This was observed for all incubations. 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 60 70 80 90 100 110 120 % Remaining Time (min.) Female Human EGBE Clearance, % Remaining Trial 1 Trial 2
53 The heat deactivated negative control worked as expected wi th the percent remaining staying around 100% for all incubations meaning that boiling the cells effectively inhibited enzymatic metabolism. This was performed for all the trials. II. METABOLITES At first, a quick 2.70 minute method was used to analyze the incubation supernatants from ethylene glycol 2 ethy lhexyl ether and ethylene glycol monohexyl ether The 2.70 minute method began with 95% water solvent A and 5% methanol solvent B. A linear gradient to 100% B occurred over 0.50 minutes, followed by an isocratic gradient of 100% B for 1.20 minutes. A quick linear gradient back to 95% solvent A occurred over 0.10 minutes and remained at 95% solvent A for 0.90 minutes to equilibrate column for subsequent run s After analyzing incubation supernatants with this method, a peak with a retention time of 1.46 minut es appeared close to the expected retention time of 1.50 minutes of ethylene glycol 2 ethylhexyl ether as incubation times increased, as shown in Figure 3 10. This was inconvenient because the unexpected peak interfered with properly integrating the area under the peak of ethylene glycol 2 ethylhexyl ether. Since the peak increased as incubation time s increased, it was suspected that a metabolite was responsible for this peak. T he u nknown metabolite had to enter Q1 with a mass of 175.0 Da and Q3 with a mass of 63.0 Da. This meant that initial fractioning of the metabolite had to occur before entering Q1.
54 Figure 3 10. Retention peak of ethylene glycol 2 ethylhexyl ether (1.50 minutes) and suspected metabolite (1.46 minutes left hand peak ) from incubation with male mouse hepatocytes The suspected metabolite peak increases as the incubation time increases. 5 minutes 1 5 minutes 30 minutes 60 minutes 120 minutes
55 Figure 3 11. Suspected ethylene glycol 2 ethylhexyl ether glucuronide metabolite. Glycosidic bond breakage is shown. The glucuronide metabolite was chosen as possible candidate for the unknown 1.46 minute peak. Fragmentation of the glycosidic bond has been observed when exposed to high declustering potentials. The structure of the suspected metabolite and bond break are shown in Figure 3 11. To get better separation from the two retention peaks shown in Figure 3 10, the 2.70 minute chromatography method was extended to the 5.70 minute method described in the methods section. A longer linear gradient from 5% B to 100% B would improve separation. In addition, the mass of the glucuronide conjugate was added for detection. This was the protonated form of the suspected ethylene glycol 2 ethylhexyl ether glucuronide meta bolite with a Q1 mass of 351.0 Da and a Q3 m ass of 63.0 Da. The declustering potential was also set to zero for the 351.0/63.0 Q1/Q3 detection to prevent unwanted fragmentation at the glyc osidic bond before entering Q1 The longer chromatography method resulted in better separation of the peaks, as Figure 3 12 shows. This enabled better integration of the ethylene glycol 2 ethylhexyl ether peak. The suspected metabolite peak clearly increa sed as incubation time increased while that of ethylene glycol 2 ethylhexyl ether decreased. Furthermore, the 351.0/63.0 Q1/Q3 mass showed that the interfering peak correspond ed to the conjugated glucuronide metabolite of ethylene glycol 2 ethylhexyl ether, as Figure 3 13 shows. To the best of my knowledge, this is the first time the ethylene glycol 2 ethylhexyl ether glucuronide
56 Figure 3 12. Detection peaks of ethylene glycol 2 ethylhexyl ether (retention time of 3.26 minutes) and the suspected metabolite (retention time of 3.08 minutes left hand peak ). The longer 5.70 minute method was able to better separate the peaks compared to the 2.70 minute method shown in Figure 3.10. These spectra originated from male mouse hepatocytes incubation with Q1/Q3 masses of 175.000/63.000 Da 5 minutes 1 5 minutes 30 minutes 60 minutes 120 minutes
57 Figure 3 13. Spectra of male mouse hepatocytes incubations when scanning for the ethylene glycol 2 ethyl hexyl ether glucuronide metabolite with Q1/Q3 masses of 351.000/63.000 and a declustering potenti al of zero. 5 minutes 1 5 minutes 30 minutes 120 minutes 60 minutes
58 conjugate has been reported. The ethylene glycol 2 ethylhexyl ether glucuronide conjugate was detected in all hepatocyte incubations. The same method for detecting the glucuronide metabolite was applied for ethylene glycol monohexyl ether and ethylene glycol monobutyl ether. The Q1/Q3 masses used were 323.100/63.000 Da and 295.100/62.900 Da for ethylene glycol monohexyl ether and ethylene glycol monobutyl ether, respectively. The declustering potential was also set to zero for this analysis. H owever, a retention peak for the glucuronide metabolite for these two comp o unds was not detected.
59 CHAPTER 4 DISCUSSION I. METABOLIC CLEARANCE Ethylene glycol 2 ethylhexyl ether had a shorter half life than ethylene glycol monohexyl ether i n all incubations, even when large standard errors from some of the ethylene glycol monohexyl ether incubations were taken into account. The glucuronide metabolite of ethylene glycol 2 ethylhexyl ether suggests that transport through the hepatocyte cell m embrane is important to predict metabolic clearance since the chemical has to first penetrate the cell membrane Alcohol dehydrogenase and aldehyde dehydrogenase, p hase I metabolism enzymes are found in the cytoplasm of hepatocytes (Buehler et al., 1982; Maeda et al., 1988) T he phase II metabolism enzyme UDP glucuronosyltransferase is responsible for glucuronidation of xenobiotics (Chowdhury et al., 1985). The mechanism of glucuronidation is shown in Figure 4 1. The mechanisms by which the three glycol ethers pass th rough the cell membrane are un known. Burton et al. (1996) mention ed that transcellular permeability of a chemical is a function of complex physicochemical properties such as size, lipophilicity, hydrogen bond potential, charge, and conformation. The amount of chemical the cells are exposed to should also affect transcellular permeability. In this experiment, cells were exposed to 1 M of each test article as required by t he National Toxicology Program. However, higher concentrations may be desirable for metabolite screenings.
60 Figure 4 1. Glucuronidation mechanism of hydroxyl groups by UDP glucuronosyltransferase (Patana, 2009) In a series of chemicals, like the gly col ethers used in this study, one of these factors like lipophilicity, may dominate the others. Hansch et al. (1995) report ed that the octanol/water log partition coefficient of ethylene glycol monobutyl ether is 0.83 while that of ethylene glycol monohe xyl ether is 1.57 (Centers for Disease Control and Prevention, 2004) The higher octanol/water partition of ethylene glycol monohexyl is expected since the extra ethyl carbon chain would increase solubility in octanol. Using this same reasoning, the octano l/water partition coefficient of ethylene glycol 2 ethylhexyl ether is expected to be higher than 1.57. This also suggests that the lipophilicity of ethylene glycol 2 ethylhexyl ether is higher than the smaller glycol ethers investigated. A higher rate of passive diffusion through the cell membrane by ethylene glycol 2 ethylhexyl ether could explain why its half life is shorter than ethylene glycol monohexyl ether. If this is the case, the half life of ethylene glycol monobutyl ether would be expected to be even longer. For example, the half lives of female and male mouse incubations were longer for ethylene glycol monohexyl ether than for ethylene glycol 2 ethylhexyl ether, as Figures 3 4 and 3 5 shows. Chemicals with longer half lives would be expected to be more damaging to organisms since they can circulate for a
61 longer period of time and interact with other cells, while those with short half lives should be excreted faster. Unfortunately, the half life for the incubations with ethylene glycol monobutyl ether could not be calculated. Figure 3 8 shows that the percent remaining reaches over 100% at the later time points suggesting that more of the compound is present than initially added. As mentioned earlier, it is possible that an endogenous compound in the hepatocytes with the same retention time and fragmentation pattern is interfering with the detection of ethylene glycol monobutyl ether, as the no compound negative control showed. It is also possible that a metabolite is interfering with the detectio n peak as the ethylene glycol 2 ethylhexyl ether glucuronide metabolite initially did with the short 2.70 minute method. The scan for the glucuronide metabolite of ethylene glycol monobutyl did not yield a detection peak. Therefore, a different metabolite may be respon sible that should be investigated If a metabolite is responsible for the observed increase in the percent remaining, the n some basic properties can be predicted The increase in percent remaining over 100% signifies that the metabolite is ion ized as or more readily than et hylene glycol monobutyl ether Since the mobile phases have 0.1% formic acid by volume, the met abolite is likely basic and is protonated at a faster rate than the glycol ether. The detection of all metabolites for a given x enobiotic is arguably the most challenging objective of metabolomics. Radiolabeling techniques make it easier to detect metabolites that exhibit a specific isotope signal. However, this comes at a cost to the a safety pre cautions Not using isotope labels leaves us with two options to detect metabolites. The method used to detect the ethylene glycol 2
62 ethylhexyl ether glucuronide metabolite can be employed to detect other metabolites. However, this requires previous knowle dge of enzymatic changes to xenobiotics and limits the possibility of finding new enzymatic changes. We can also use an open scan to detect all chemicals left in the supernatant of the incubations. This spectrum could then be normalized to the no compound negative control to determine what peaks a re due to the added glycol ether This second method would ideally give separated peaks with each corresponding to different metabolites and the remaining unmetabolized glycol ether. A long linear gradient may help ensure that each peak is completely separated from other s The difficult task would then be to assign the correct metabolite to each peak. Moreover, the properties of the mobile phases would have to be changed to detect metabolites that have an acidic h ydrogen that would not be detected with the conditions used in this experiment. The formic acid used in the mobile phases was used to help ionize the glycol ethers by protonation. Metabolites with acidic hydrogens would have to be deprotonated to be ionize d. Therefore, a base would have to be added to the mobile phases to help deprotonate acidic hydrogens For example, the 2 butoxyaceticacid metabolite of ethylene glycol monobutyl ether would have a stronger detection peak if a basic mobile phase is used in stead of an acidic mobile phase since it is a carboxylic acid and deprotonated more than it would be protonated in acidic media. II. CRYOPRESERVED HEPATOCYTES GOOD METABOLISM MODELS? Hepatocytes have been investigated as models to predict in vivo hepatic metabolic clearance. McGinnity et al. (2004) used fresh hepatocytes to investigate similarities in metabolites and clearance compared to animal studies. They observed that there was a
63 statistically significant correlation between the scaled metabo lic clearance of fresh human hepatocytes and test subjects. The data from the fresh human hepatocytes was then used to compare to commercially available cryopreserved hepatocytes like the ones used in this study. Fourteen drugs metabolized by the major hum an cytochromes P450 and uridine diphosphate glucuronosyltransferases were used to analyze the activity of freshly isolated and cryopreserved human and dog hepatocytes. It was determined that the cryopreserved hepatocytes retained on average 94% and 81% of the intrinsic clearance determined in fresh cells from humans and dogs, respectively. Furthermore, cryopreserved hepatocytes could retain their full activity for more than one year in liquid nitrogen. Cryopreserved hepatocytes, as determined by McGinnity et al. (2004), appears to be good models for metabolism studies. But how can they b e improved to better emulate the metabolic processes in vivo ? The hepatocytes used in this study were cultured in suspension. Yet, liver cells in vivo are attached to each o ther creating a three dimensional network that is not replicated in suspensions. Brophy et al. (2009) investigated this possible problem by using a novel rocking technique that induced spheroid formation. Freshly isolated rat hepatocytes were cultured usin g either rocked or rotational motion. After 24 hours of incubation, 85% of inoculated hepatocytes using the rocked technique had formed into spheroids greater than 40 m in diameter. In contrast, only 58% of hepatocytes incorporated into spheroids using th e rotational technique and those that were unattached appeared dead. Brophy et al. (2009) proceeded to test differences in gene expression between the rocked hepatocytes and fresh hepatocytes. This was done by creating a custom
64 microarray of 242 liver re lated genes with rat specific oligonucleotide sequences. The rat hepatocytes rocked over 14 days showed that average expression of 206 genes remained stable relative to the baseline. However, expression increased 2 fold or greater in 11 genes and decreased 50% or more in 25 genes. Thirty nine genes involved in phase I metabolism were included in the microarray and showed the greatest variability of any group of genes tested. Only the average expression of 19 phase I genes remained stable over 14 days One p hase I gene was up regulated while the expression of another declined. Eighteen other phase I genes were down regulated less than 50% of baseline. Expression of 14 of 16 phase II metabolism genes remained stable during the 14 days of rocked spheroid forma tion. All five UDP glucuronosyltransferases genes remained stable. Brophy et al. (2009) suggest that spheroid formation may better replicate the in vivo properties of hepatocytes. III. FUTURE DIRECTIONS AND CONCLUSION There is no question that the half life standard errors of the glycol ether incubations must be reduced before any definitive conclusions can be made about metabolic clearance differences among species and gender incubated with the glycol ethers in this experiment. The small standard e rror of the positive control 7 ethoxycoumarin hints that the current method and instrument to detect the glycol ethers may not be adequate. It seems clear that the half life of ethylene glycol 2 ethylhexyl ether is shorter than that of ethylene glycol mono hexyl ether. The half life of ethylene glycol monobutyl ether was not calculated and future experiments should focus on determining
65 if an endogenous compound and/or metabolite is responsible for the observed increases in percents remaining as incubation ti me increases. Future experiments should also be performed in triplicate trials for better statistical analysis The National Toxicology Program requested that the hepatocyte incubations be performed with a final hepatocyte concentration of 1 million viab le cells per milliliter. However, I would try to persuade the National Toxicology Program to allow the incubations with 500,000 or 750,000 viable cells per milliliter to reduce costs and to prevent some comp ounds from being metabolized quickly, as was the case with the positive control 7 ethoxycoumarin. Finally, metabolite screening remains a major challenge with chemicals that are not very acidic or basic like the glycol ethers used in this study Quantitation methods that can detect metabolites that form b oth negative and positive ions must be created. These methods should have long linear gradients to effectively separate all peaks from each other even though this will increase the length of the experiment significantly. In conclusion, the half life st andard errors of the incubations make it difficult to determine which metabolic clearance more closely resembles that of humans It is important to determine which animal model more closely resembles human metabolism so that the effect of these glycol ethe rs can be studied and accurately correlated to possible human effects. Animal studies of ethylene glycol monobutyl ether and smaller glycol ethers suggest that glycol ethers may have detrimental effects in humans.
66 APPENDIX A PARAMETERS AND SAMPLE SPE CTRA OF EFFECTS Operators Manual which can be found online (see Applied Biosystems, 2005, in references). Source Dependent Parameters. Optimal source dependent parameter values depend on the liquid chromatography conditions. Source dependent parameters should be optimized at or near the desired liquid chromatography flow conditions GS1 (Gas 1): The GS1 parameter controls the nebulizer gas. The nebulizer gas helps generate the small dro plets of sample flow and affects the spray stability and sensitivity. GS2 (Gas 2): The GS2 parameter controls the auxiliary, or turbo gas in the TurboIon Spray (TSI) probe used in electrospray ionization conditions. Using an APCI probe, this parameter sho uld not be used (Applied Biosystems, 2011). TEM (Temperature): The TEM parameter controls the temperature of the heater from the APCI probe. It is used to help evaporate the solvent to produce gas phase sample ions. which flows high as possible without los ing sensitivity. IS (IonSpray Voltage): The IS parameter controls the voltage applied to the needle that ionizes the sample in the source. It depends on the polarity, and affects the stability of the spray and the sensitivity. Ihe (Interface Heater): The i he parameter switches the interface heater on and off. Heating the interface helps maximize the ion signal and prevents contamination of the ion
67 heated to 100C. NC (Nebulizer, or Needle Current): THE NC parameter controls the current applied to discharge ionizes the solvent molecules, which in turn ionize the sample molecules. Compound Dependent Pa rameters. The available compound dependent parameters consist mostly of lens elements in the ion path. Optimal values for compound dependent parameters do not depend on the liquid chromatography flow conditions ; therefore, the parameters can be optimized u sing any sample introduction technique. The parameters listed here are generally the only ones that need to be optimized. CAD (CAD Gas): The CAD parameter controls the pressure of collision gas in the collision cell, Q2 (refer to Figure 2 1), during MS/MS scans. The collision gas acts as a target to fragment the precursor ions. When the parent ions collide with the collision gas, they can dissociate to fragment ions. DP (Declustering Potential): The DP parameter controls the voltage on the orifice, It is used to minimize the solvent clusters that may remain on the sample ions after they enter the vacuum cham ber, and, if desired, to fragment ions before entering the QJet Ion Guide. The higher the voltage, the higher the energy imparted to the ions. If the DP parameter is too high, unwanted fragmentation may occur.
68 EP (Entrance Potential): The EP parameter con trols the DC potential of the combination of Q 0 the QJet Ion Guide, and the lens between the QJet Ion Guide and Q0. The same DC potential is applied to Q0, the lens and the Qjet Ion Guide, and is equal to EP parameter. CXP (Collision Cell Exit Potentia l): The CXP parameter controls the collision cell exit potential, which is used to focus and accelerate the ions out of the collision cell, Q2. CXP is the potential difference between RO2 and ST3 (the stubby lens between Q2 and Q3) and is not mass dependen t RO2 (Collision Cell Rod Offset): The RO2 parameter is also referred to as the Collision Energy (CE). This parameter controls the potential applied to the collision cell, Q2. In MS/MS scans, CE is the potential difference between Q0 and Q2. This is the amount of energy that the precursor ions receive as they are accelerated into the Q2 collision cell, where they collide with gas molecules and fragment. IE1 (Ion Energy 1): The IE1 parameter controls the potential difference between Q0 and RO1. Although t his parameter does affect the sensitivity, it has a greater impact on the resolution of the peaks, that is, peak shape, and is considered a resolution parameter. IE3 (Ion Energy 3): The IE3 parameter controls the potential difference between RO2 and RO3. A lthough this parameter does affect sensitivity, it has a greater impact on the resolution of the peak shape, and is considered a resolution parameter. IQ1 (Focusing Lens 1): This is the voltage on the first inter quadrupole lens and is dependent on the ent rance potential. IQ2 (Focusing Lens 2): IQ2 = Q0 + offset. Set the offset between 8 and 30 V for positive ions and between 8 and 30 V for negative ions.
69 Positions of Q0, RO2 and ST3, mentioned above, are shown in Figure A 1. Figure A 1. Schematic of the API are shown.
70 Figure A 2. Fragmentation pattern of (A) ethylene glycol monobutyl ether and (B) ethylene glycol monohexyl ether when Collision Enery is 15 V.
71 Table A 1. API 5000 Triple Quadrupole LC/MS/MS analyte parameters.
72 APPENDIX B ADDITIONAL GLYCOL ETHER INFORMATION I. ETHYLENE GLYCOL MONOBUTYL ETHER There are two common methods for synthesizing 2 butoxyethanol. The first involves the reaction of ethylene oxide with anhydrous butyl alcohol in the presence of a catalyst (Hazardous Substances Data Bank, 2012; UNEP, 1997; US Department of Health and Human Services, 1998). When this method is used, other glycol ethers are also produced such as dieth ylene glycol and diethylene glycol monobutyl ether (DOW, 1958). The second method employs the direct alkylation of 2 chloroethanol or ethylene glycol using sodium hydroxide and an alkylating agent such as dibutyl sulfate (US Department of Health and Human Services, 1998). Absorption and Distribution Studies of skin absorption of ethylene glycol monobutyl ether have not lacked controversy. Johanson and Boman (1991) compared the total skin absorption of ethylene glycol monobutyl ether by mouth only and body only exposures using four male volunteers. The four male volunteers were exposed to 50 ppm 2 butoxyethanol mouth only for 2 hours, followed by 1 hour of no exposure, and then exposed in a chamber of 50 ppm body only for 2 hours while breathing fresh air th rough a respirator. Finger prick blood samples were taken periodically for analysis of 2 butoxyethanol under the assumption these blood samples represented mixed arterial blood. Johanson and Boman (1991) concluded that about 75% of the total uptake of 2 bu toxyethanol was due to skin absorption with the area under the curve for body only exposure being three to four fold greater than mouth only exposure. Corley et al. (1994) were skeptical of these results and argued that physiological differences like rela tive surface area, blood perfusion, and barrier thickness should favor the absorption of 2
73 and assuming that the finger prick blood sample was a suitable representation of venous blood draining the skin, Corley et al. (1994) concluded that dermal uptake should not contribute to more than 22% of the total uptake of 2 butoxyethanol. To further investigate the absorption of 2 butoxyethanol, Corley et al. (1997) conducted their own studies to determine if the prick blood sample method Johanson and Boman (1991) used were representative of systemic arterial blood. In their study, Corley et al. (1997) exposed one arm of their human subjects to 50 ppm [ 13 C] 2 butoxyethanol for 2 hour s. Blood samples from the exposed arm were collected through the finger prick procedure while cathethers were installed in the antecubital vein of the unexposed arm. The assumption by Johanson and Boman (1991) that finger prick blood samples were represent ative of systemic arterial blood was disproven when Corley et al. (1997) showed that the concentration of 2 butoxyethanol was about 1,500 fold higher in the finger prick blood samples than the samples from the unexposed arm. This clearly showed that the si te of entry of 2 butoxyethanol was important to determine how it was distributed. Most importantly, Corley et al. improved the physiologically based pharmacokinetic model that Johanson and Boman were used.
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