CHARACTERIZATION OF THE INHIBITION OF GENISTEIN
GLUCURONIDATION BY BISPHENOL A IN HUMAN AND RAT
LIVER MICROSOMES
By
JANIS LAURA COUGHLIN
A thesis submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey and
The Graduate School of Biomedical Sciences
University of Medicine and Dentistry of New Jersey
in partial fulfillment of the requirements for the
degree of Master of Science
Joint Graduate Program in Toxicology
written under the direction of
Dr. Brian Buckley
and approved by
______
______
______
______
New Brunswick, New Jersey
OCTOBER 2011 ABSTRACT OF THE THESIS
Characterization of the Inhibition of Genistein Glucuronidation by Bisphenol A
in Human and Rat Liver Microsomes
By JANIS LAURA COUGHLIN
Thesis Director: Dr. Brian Buckley
Genistein is a natural phytoestrogen that is found abundantly in the soybean.
Bisphenol A (BPA) is a synthetic chemical used in the synthesis of polycarbonate plastics
and epoxy resins. Endocrine disrupting properties of both genistein and BPA have been
well established by various laboratories. Because the adverse biological effects caused
by genistein and BPA are similar, and may include common co-exposure scenarios in the
general population such as in the consumption of a soy-milk latte from a polycarbonate
plastic coffee mug, analysis of the perturbation of the metabolism via glucuronidation of
genistein in the presence of BPA has been assessed. Human and rat liver microsomes
were exposed to varying doses of genistein (0 to 250 μM) in the absence (0 μM) or
presence (25 μM) of BPA. Treatment with 25 μM BPA caused non-competitive
inhibition of the glucuronidation of genistein in human liver microsomes with a Ki value of 58.7 μM, represented by a decrease in Vmax from 0.93 ± 0.10 nmol/min/mg in the
ii absence of BPA to 0.62 ± 0.05 nmol/min/mg in the presence of BPA, and a negligible change in Km values between treatment groups. The addition of BPA to incubations performed in rat liver microsomes resulted in competitive inhibition of the glucuronidation of genistein at a Ki of 35.7 μM; Vmax values remained steady (2.91 ± 0.26 nmol/min/mg in the absence of BPA and 3.05 ± 0.41 nmol/min/mg in the presence of
BPA), while Km values increased in the presence of BPA (49.4 ± 14 μM in the absence of
BPA and 84.0 ± 28 μM in the presence of BPA). These findings indicate that the type of inhibition on genistein glucuronidation exerted by BPA differs among species.
iii ACKNOWLEDGEMENTS
I would like to thank a handful of people who have supported me in various ways throughout my studies and research. Many thanks to my mentor Dr. Brian Buckley, who graciously welcomed me into his lab with open arms. I am incredibly fortunate to have had the opportunity to work for such a wonderful advisor. He has taught me so much, both professionally and personally, and has always greeted me with a smile. I sincerely appreciate that he always made time for my questions and concerns, regardless of how unimportant or trivial they may have been. I am incredibly grateful for his unparalleled mentoring and guidance.
Special thanks are extended to my committee members, Dr. Kenneth Reuhl, Dr.
Lauren Aleksunes, and especially Dr. Paul Thomas for his tremendous patience and the countless hours he spent with me designing experiments, checking calculations, and discussing and analyzing results of the metabolism experiments. I truly appreciate him opening his lab to me so freely, and for being such a great example of not only a phenomenal scientist, but also a wonderful person.
A big “thank you” to the past and present members of the Buckley lab, especially
Dr. Bozena Winnik for her tireless assistance in the method development, Dr. Hilly Yang for his technical assistance and troubleshooting of the HPLC-MS/MS, and Dr. Elizabeth
McCandlish for her chemistry expertise.
I greatly appreciate the financial support provided by the National Institutes of
Health (Grant Number T32ES007148), National Institute of Environmental Health
iv Sciences (Grant Number ES05022), and the Environmental and Occupational Health
Sciences Institute.
A special thank you to Michal Sheleg, who somehow made studying fun, and
always made lunchtime quite cheerful!
A big mahalo to my parents, who have generously supported me in so many ways.
I have no idea how to even begin to repay them.
Merci un mille fois to Stu, whose unwavering support and unfaltering belief in me
gave me the courage to follow my heart, and had made all the difference in my life.
Heaven only knows where I would be without him.
v TABLE OF CONTENTS
Page
ABSTRACT OF THE THESIS…………………………………………... ii
ACKNOWLEDGEMENTS…………………………………………...….. iv
LIST OF TABLES………………………………………………………… ix
LIST OF FIGURES……………………………………………………….. x
LIST OF ABBREVIATIONS…………………………………………….. xi
1.0. GENERAL INTRODUCTION………………………………………. 1
1.1. Endocrine Disrupting Compounds...…………………………. 1
1.2. Glucuronidation………………………………………………. 3
1.2.1. Enzyme Inhibition………………………………….. 4
1.3. Genistein……………………………………………………… 7
1.3.1. Affinity for the estrogen receptor……….…….…… 8
1.3.2. Biological effects………………………………...…. 8
1.3.3. Pharmacodynamics and pharmacokinetics……...….. 11
1.3.4. Environmentally relevant exposure levels……..…… 12
1.4. Bisphenol A…………………………………………..………. 14
1.5. Rationale for Analysis of Genistein and BPA………………… 17
1.5.1 Comparable metabolism…………………………….. 17
1.5.2. Similar biological effects…………………………… 18
1.5.3. Common exposures…………………………………. 19
2.0. AIM ONE: DETERMINATION VIA SPE AND HPLC-MS/MS…... 20
2.1. Abstract………………………………………………...…….. 20
vi 2.2. Introduction…………………………………………….……. 21
2.3. Materials and Methods………………………………….…… 25
2.3.1. Solid phase extraction……………….……..……… 26
2.3.2. Standard solutions…………………….…………… 26
2.3.3. Blank controls………………………….………….. 27
2.3.4. Chromatographic conditions………………………. 27
2.3.5. Mass spectrometry…………………………………. 28
2.3.6. Statistical analyses…………………………………. 29
2.4. Results………………………………………………...……... 30
2.4.1. Retention times, limits of detection, and calibration
curves……………………………………………….. 30
2.4.2. Recoveries………………………………………….. 30
2.4.2.1. Bond Elut Plexa SPE cartridges………….. 31
2.4.2.2. Oasis HLB SPE cartridges………………... 31
2.4.2.3. UCT C18 SPE cartridges…………………. 31
2.4.2.4. Glucuronide recoveries……………...……. 32
2.4.3. HPLC column performance………………………… 32
2.5. Discussion…………………………………………………….. 34
2.6. Conclusion……………………………………………………. 37
3.0. AIM TWO: INHIBITION OF GLUCURONIDATION…………….. 38
3.1. Abstract………………………………………………...…….. 38
3.2. Introduction…………………………………………….……. 40
3.3. Materials and Methods………………………………….…… 43
vii 3.3.1. Glucuronide formation……………….……..……… 43
3.3.2. Inhibition studies…………………..….…………… 44
3.3.3. Sample preparation...…………………….…..…….. 44
3.3.4. Standard solutions…………………………………. 45
3.3.5. Blank controls…..…………………………………. 45
3.3.6. Analytical conditions………………………………. 45
3.3.7. Data analyses………………………………………. 46
3.4. Results………………………………………………...……... 49
3.4.1. BPA glucuronidation kinetics……………………… 49
3.4.2. Genistein glucuronidation kinetics……………..….. 49
3.5. Discussion…………………………………………………….. 51
3.6. Conclusion……………………………………………………. 54
4.0. GENERAL DISCUSSION…………………………………………… 55
4.1. Areas of further research……………………………………… 59
5.0. REFERENCES………………………………………………………... 61
viii LIST OF TABLES
1.1. Estrogenic potentials of 17 β-estradiol, genistein, and BPA………………... 70
2.1. Summary of mass spectrometric parameters………………………………… 71
2.2. Retention times of analytes using Kinetex C18 and Discovery C8..………… 72
ix LIST OF FIGURES
1.1. Chemical structures of genistein and genistein gluc…………….…………… 73
1.2. Chemical structure of 17 β-estradiol…………………………………………. 74
1.3. Chemical structures of BPA and BPA gluc………..…………….…………… 75
1.4. Genistein and BPA co-exposure scenario……………………………………. 76
2.1. Chromatograms and mass spectra using Kinetex C18………………………. 77
2.2. Chromatograms and mass spectra using Discovery C8…..…………………. 78
2.3. SPE recoveries of BPA and genistein………………………………….…….. 79
2.4. Recoveries of analytes from optimized SPE conditions……………………… 80
3.1. Optimization of HLM protein concentration………………………………… 81
3.2. Optimization of RLM protein concentration………………………………… 82
3.3. BPA glucuronidation kinetics………………………………………………… 83
3.4. Genistein glucuronidation kinetics in HLMs, pooled data……………………. 84
3.5. Genistein glucuronidation kinetics in RLMs, pooled data……………………. 85
3.6. IC50 of BPA for genistein glucuronidation in HLMs ……………………….. 86
3.7. Genistein glucuronidation kinetics in HLMs, replicate 1 ……………………. 87
3.8. Genistein glucuronidation kinetics in HLMs, replicate 2 ……………………. 88
3.9. Genistein glucuronidation kinetics in HLMs, replicate 3 ……………………. 89
3.10. Genistein glucuronidation kinetics in RLMs, replicate 1 …………….……. 90
3.11. Genistein glucuronidation kinetics in RLMs, replicate 2 ………….………. 91
3.12. Genistein glucuronidation kinetics in RLMs, replicate 3 ……………..……. 92
x LIST OF ABBREVIATIONS
BPA bisphenol A
BPA gluc bisphenol A β-D-glucuronide
EDC endocrine disrupting compound
EA 3 mL ethyl acetate and 3 mL acetonitrile
EM 3 mL ethyl acetate and 3 mL methanol
EMA 3 mL ethyl acetate, 2 ½ mL methanol, and 2 ½ mL acetonitrile
ESI electrospray ionization genistein gluc genistein 4’-β-D-glucuronide
HLB hydrophilic-lipophilic balance
HLM human liver microsomes
HPLC high performance liquid chromatography
HPLC-MS/MS high performance liquid chromatography tandem mass spectrometry
MA 3 mL methanol and 3 mL acetonitrile m/z mass-to-charge ratio
RBA relative binding affinity
RLM rat liver microsomes
RSD relative standard deviation
SPE solid phase extraction
SRM selective reaction monitoring
xi UCT United Chemical Technologies
UDPGA uridine 5’-diphospho-glucuronic acid
UGT uridine 5’-diphospho-glucuronosyltransferase
Vmax maximal reaction rate
xii 1
1.0. GENERAL INTRODUCTION
1.1. Endocrine Disrupting Compounds
Endocrine disrupting compounds (EDC) are chemicals that perturb the endocrine system by altering the binding, release, or metabolism of endogenous hormones. EDCs mimic endogenous estrogens and androgens and may act as either agonists or antagonists at sex hormone receptors, thereby affecting physiology and behavior that are normally under the regulation of sex steroid hormones.
One of the most infamous EDCs is diethylstilbestrol (DES), a synthetic estrogen that was prescribed to pregnant women from approximately 1940 to 1970 to prevent miscarriages. Although mothers treated with DES show a modest increase in susceptibility to breast cancer, the primary pathological effects of DES treatment are observed in the offspring of the DES-treated mothers. Daughters born to mothers who took DES during pregnancy exhibit a significantly increased incidence of otherwise rare vaginal clear cell adenocarcinomas (Verloop et al., 2010), and sons of DES-treated mothers exhibit develop testicular cysts, hydrospadias, and/or cryptorchidism (Palmer et al., 2009). The latent, trans-generational effects of DES first observed around 1950 dramatically raised awareness of the potential for compounds to alter endocrine function in individuals exposed at critical developmental time periods as well as in subsequent generations.
Endocrine disruption is not caused solely by pharmacological agents; many environmental and dietary compounds have been shown to alter the endocrine system as well. Endocrine disrupting properties have been reported for various classes of 2 chemicals, including naturally-occurring phytoestrogens (genistein, daidzein, biochanin
A) (Casanova et al., 1999; Peterson and Barnes, 1993); commonly used pesticides and herbicides (e.g., methoxychlor, atrazine) (Armenti et al., 2008); anti-bacterial agents used in deodorant, soap, and toothpaste (e.g., triclosan) (Dann and Hontela, 2011); synthetic plasticizers (e.g., bisphenol A, phthalates) (Richter et al., 2007; Rubin et al., 2001;
Hannas et al., 2011); and flame retardants (e.g., polybrominated diphenyl ethers) (Perez et al., 1998). Organisms are exposed to an assortment of diverse chemicals through various media and routes of exposure over the course of their lifetimes, introducing them to innumerable potential EDCs. Individuals at different stages of development are differentially affected by the adverse effects of EDCs. In general, exposure to EDCs during the critical windows of gestation and neonatal development result in the most pronounced effects of endocrine disruption (Patisaul and Polston, 2010). Inactivation of compounds via metabolic routes such as glucuronidation can impact the effects exerted by EDCs. Inhibition of detoxification pathways such as glucuronidation can result in enhanced toxicity of xenobiotics.
3
1.2. Glucuronidation
Upon absorption, xenobiotics are generally biotransformed into metabolites with increased water solubility compared to the original xenobiotic in order to facilitate urinary excretion of the compound from the body. The polar metabolites are usually less biologically active than their parent compounds, with a notable exception being morphine-6-glucuronide (Osborne et al., 1999). One of the primary forms of phase II xenobiotic metabolism is glucuronidation, in which uridine 5’-diphospho- glucuronosyltransferase (UGT) enzymes transfer the glucuronosyl group from the required co-factor uridine 5’-diphospho-glucuronic acid (UDPGA) to a substrate, thus catalyzing the synthesis of a β-D-glucuronide conjugate. UGTs are promiscuous enzymes capable of conjugating a wide variety of structurally diverse substrates by attacking their substrates at nucleophilic sites containing oxygen (ex: hydroxyls, carboxylic acids), nitrogen (ex: amines), sulfur (ex: thiols), and occasionally carbon.
There are currently 17 known human UGT isoforms (Mackenzie et al., 2005), which are subdivided into two main families, UGT1 and UGT2, based on alternative splicing of exons. UGTs are well conserved among species; however, several specific isoforms have diverged in both structure and function among inter-species homologues.
For example, the predominant UGT responsible for the glucuronidation of bisphenol A in the rat is UGT2B1, which is homologous to human UGTs 2B7 and 2B17, neither of which is the primary UGT involved in BPA glucuronidation in humans (Elsby et al.,
2001). Instead, human UGT2B15 is the key enzyme involved in BPA metabolism
(Hanioka et al., 2008). Humans and rats have several orthologous UGTs, including 1A1,
1A3, 1A5, 1A6, 1A7, 1A8, and 1A10 (Tukey and Strassburg, 2000). Humans, but not 4 rats, also have functional UGTs 1A4 and 1A9 (Mackenzie et al., 2005). In general, rodents have a greater overall capacity for glucuronidation as compared to primates
(Elsby et al., 2001).
UGTs are distributed throughout the body in an organ-specific manner in organs including the kidney, lung, intestine, and brain. Of all organs, the liver contains the most
UGTs, which are specifically located along the smooth endoplasmic reticulum. There exist a few UGT isoforms that are unique to extra-hepatic tissues, such as human isoforms UGT 1A7 and 1A10 (Tukey and Strassburg, 2000; Miners et al., 2006).
In vitro glucuronidation of different substrates may be assessed by taking advantage of the innate metabolic capabilities of liver microsomes. Since the active site of UGT enzymes is located along the luminal membrane of the smooth endoplasmic reticulum, pores must be formed in order to perforate the endoplasmic reticulum membrane, allowing both the substrate and co-factor UDPGA to access the catalytic site of the UGT. Alamethicin is a commonly used pore-forming agent in such in vitro assays because it does not alter the Km of the enzymes (Kilford et al., 2009). Additionally,
alamethicin has been shown to not disrupt cytochrome p450 activity, unlike detergents
such as Brij 58 (Fisher et al., 2000).
1.2.1. Enzyme Inhibition
Glucuronosyltransferases, like other enzymes, are subject to inhibition. External
factors such as temperature and pH can influence enzyme inhibition, as can the presence
of xenobiotics. Certain substrates can interact with enzymes in different ways that can 5 cause inhibition of an enzyme’s activity, which can directly alter xenobiotic metabolism.
Nonspecific inhibitors often result in the denaturation of the enzyme’s protein, which can be caused by extreme temperatures or changes in pH. Many xenobiotics, however, are specific inhibitors that alter the activity of enzymes. Competitive inhibitors are usually structurally similar to the enzyme’s endogenous substrate, and thus can compete for the enzyme’s active site. When bound to the enzyme’s active site, competitive inhibitors render the enzyme inactive. Competitive inhibition is often reversible via displacement with sufficiently high substrate concentrations; therefore, the level of competitive inhibition is determined by both inhibitor concentration and substrate concentration, as well as their relative affinities to the enzyme. Hallmarks of competitive inhibition are increasing Km values and unchanging maximal reaction velocity (Vmax) values in the
presence of increasing inhibitor concentrations. Because the effect of the inhibitor can be
overcome by introducing higher substrate concentrations, the reaction may achieve the
same Vmax both with and without inhibitor. In order to achieve this same Vmax, substrate concentrations must be increased in the presence of inhibitor. Km is the substrate
concentration required to achieve half maximal reaction velocity. Since substrate
concentrations must be increased in the presence of inhibitor in order to overcome the
inhibition, Km must also increase. Another type of specific inhibition is non-competitive inhibition, in which inhibitors interact with enzymes at a location other than the active site. Binding of the non-competitive inhibitor to the enzyme causes a conformational change in the enzyme’s active site, subsequently preventing the substrate from being able
to bind to the enzyme. Non-competitive inhibition is often reversible, but is not
dependant upon substrate concentration. For non-competitive inhibition, Vmax values 6
decrease with increasing inhibitor concentration, while Km values remain unaffected.
Under non-competitive inhibition, the inhibitor has equal affinity for both free enzyme
and enzyme that is bound to substrate. Consequently, the binding affinity of the substrate
to the enzyme is unaffected by the presence of inhibitor, resulting in an unchanged Km under conditions of increasing inhibitor concentrations. Furthermore, since the substrate and the inhibitor do not compete for the same binding site on the enzyme, the inhibitor may not be displaced by increased substrate concentrations. As a result, the Vmax achieved with inhibitor present will be lower than that which is attainable when inhibitor is not present. Irreversible inhibition also exists when inhibitors covalently bind to an enzyme, rendering the enzyme inactive. In order for the enzyme’s function to be restored, new enzymes must be synthesized to replace the inhibited enzymes, since the enzymes cannot be reactivated once irreversibly inhibited. 7
1.3. Genistein
Genistein (4’,5,7-trihydroxyisoflavone; Figure 1.1.A) is a naturally occurring phenolic compound and the predominant phytoestrogen in many legumes and grains, with the soybean being a particularly rich source (Zhou et al., 2008). Soy products such as tofu and tempeh thereby supply dietary exposures of genistein. In addition to obvious soy products, nearly 60% of all processed foods today (i.e., meatless hot dogs and hamburgers, energy bars, cereals, cheese) contain soy, often in the form of textured soy protein or soy protein isolate, thus providing humans with a nearly constant supply of genistein from their everyday diet (Patisaul and Jefferson, 2010). Additionally, 25% of infant formula in the current US market is soy protein-based, despite very limited indications for its use (Bhatia and Greer, 2008). Genistein is found in soy products in the form of an aglycone or a glycoside, otherwise referred to as genistein glucuronide. Sales from soy-containing foods have increased from roughly $300 million in 1992 to $4 billion in 2008 due to increased demand (Patisaul and Jefferson, 2010). Soy protein has become a popular food additive because it is cholesterol- and lactose-free, as well as a good source of fiber and rich in complex carbohydrates and unsaturated fats (Patisaul and
Jefferson, 2010). Dietary supplements of genistein are used as natural alternatives to hormone replacement therapies for the relief of menopausal symptoms and serve as another source of exposure of the phytoestrogen, and often provide an average of 30 mg of genistein daily to those taking the supplements (Evans et al., 2010; Goldwyn et al.,
2000). Animals are also exposed to genistein in abundant quantities. Genistein is present in most commercially available laboratory rodent diets, usually in the form of soybean 8 meal or soy protein, at concentrations that may alter endocrine function (Thigpen et al.,
2004).
1.3.1. Affinity for the estrogen receptor
Genistein is known to have weak estrogenic activity. It is capable of binding to both estrogen receptor (ER)-α and ER-β, where it acts as either a complete agonist or an antagonist, depending upon its concentration (Shelnutt et al., 2000; Casanova et al.,
1999). Genistein has a greater affinity for ER-β than for ER-α. Compared to the endogenous ligand 17β-estradiol, genistein has a relative binding affinity (RBA) of 5.0 for ER-α and an RBA of 36 for ER-β; as well as dissociation constants (Ki) of 2.6 nM
and 0.3 nM for ER-α and ER-β, respectively (Kuiper et al., 1997;) (Table 1.1.). When
bound to the ER, genistein can upregulate the expression of estrogen-responsive genes.
The chemical structure of genistein shares structural similarity with 17β-estradiol,
specifically in regard to the presence of the phenolic ring and the physical distance
between the 4’- and 7-hydroxy groups (Figure 1.1.A and Figure 1.2.).
1.3.2. Biological effects
The interpretation of data on the pleiotropic biological effects of genistein
remains controversial. The biological effects that genistein exert may depend on age of
exposure, health status of the individual, administered dose, route of exposure, specific
composition of the individual’s intestinal microflora, and/or the presence of other dietary 9 components (Patisaul and Jefferson, 2010). Genistein has been shown to be beneficial to human health by ameliorating the unpleasant symptoms of menopause (Duncan et al.,
1999; Evans et al., 2010; Howes et al., 2006) as well as by improving bone health and reducing the risk of osteoporosis (Albertazzi, 2002). Through epidemiological data, genistein has been demonstrated to reduce the incidences of breast, prostate and colon cancers as well as cardiovascular disease by affecting cell survival and growth (Messina et al., 2006). It is a highly specific inhibitor of tyrosine-specific protein kinase activity, suggesting a possible mechanism for the anti-tumorigenic action of the phytoestrogen
(Akiyama et al., 1987). In addition to inhibiting protein tyrosine kinases, genistein also inhibits DNA topoisomerase II activity and modifies cellular differentiation (Shelnutt et al., 2000). Exposure to genistein during gestation and lactation decreases body mass and postnatal growth rate (Ball et al., 2010; Casanova et al., 1999; Wisniewski et al., 2005).
Genistein also inhibits embryonic development in vivo (Xing et al., 2010). In vitro, genistein inhibits the proliferation of human breast cancer cell line MCF-7 at an IC50 of
19 μM (Peterson et al., 1998).
Gestational and lactational exposure to genistein impairs spatial learning that
persists through adulthood, possibly by disrupting organization and development of the
hippocampus (Ball et al., 2010). Learning and memory are sexually dimorphic behaviors, and such gender-based behavioral differences are often due to organizational effects of sex hormones on brain development and morphology. Sexually dimorphic behavioral traits are sensitive to endocrine disruption and are determined by the developmental stage of the animal at the time of exposure. Genistein has also been shown to decrease aggressive behavior and increase defensive behavior in mice 10
(Wisniewski et al., 2005). Genistein also impairs immune function. Rats exposed to genistein throughout gestation and lactation exhibit increased thymic mass and long- lasting adverse immunological effects that persist through adulthood (Klein et al., 2002).
Reproductive function is adversely affected by exposure to genistein. Male mice that consume genistein exhibit atrophy of accessory sex glands, altered development of genitalia, and development of squamous metaplasia of seminal vesicles (Cline et al.,
2004; Wisniewski et al., 2005). In females, prenatal and neonatal exposure to genistein increased the incidence of abnormal multiple oocyte follicles, as well as inhibition of oocyte nest breakdown and primordial follicle assembly (Chen et al., 2007). Genistein also causes persistent demasculinization of male mice and a significant acceleration of the onset of puberty in female rodents (Wisniewski et al., 2005).
When environmentally relevant doses of genistein (less than 10 mg/kg bw/day) are administered to pregnant rats or mice during gestation and lactation, endocrine disruption of their offspring ensues, as indicated by reduced anogenital distance, disruption of estrous cycle, decreased female fertility, and reduced male copulatory ability (Wisniewski et al.; 2005, Ball et al., 2010; Klein et al., 2002). The developing fetus and neonate are particularly vulnerable and sensitive to the long-term endocrine disrupting effects of genistein (Wisniewski et al., 2005). Human data also suggest that individuals who are exposed to genistein during infancy experience precocious puberty and altered fertility (Goldman et al., 2001; Strom et al., 2001). Since genistein is transported across the placenta, developing fetuses are directly exposed to genistein via the maternal diet (Soucy et al., 2006). Prenatal and neonatal development appear to be 11 the most critical time periods for the exposure of genistein to elicit adverse health effects such as endocrine disruption (Klein et al., 2002).
1.3.3. Pharmacodynamics and pharmacokinetics of genistein
Interindividual variation in the pharmacodynamics and pharmacokinetics of genistein is determined by numerous factors including age, gender, and ethnicity (Bolling et al., 2010). The half-life of genistein in humans is approximately 3.5 hours (Cave et al.,
2007), and the average half life of genistein glucuronide in humans is 6.0 hours (Shelnutt et al., 2000).
Absorption: Genistein is readily absorbed in the gastrointestinal tract, while genistein glucuronide is usually hydrolyzed in the wall of the small intestine to its aglycone form prior to absorption in the gastrointestinal tract (Zhou et al., 2008; Kwon et al., 2007).
Distribution: Upon absorption, genistein readily distributes throughout the entire body. Genistein and genistein glucuronide can be detected in plasma within five minutes of oral administration of the parent compound, and tissues reach peak genistein concentrations approximately 80 minutes after exposure (Zhou et al., 2008).
Metabolism: Glucuronidation is the primary metabolic route for genistein in both rats and humans; the process occurs in the liver as well as in the wall of the small intestine (Andlauer et al., 2000). Glucuconidation is an effective method of detoxification of genistein, as genistein glucuronides are nearly entirely devoid of estrogenic activity with an RBA of 0.018 (Zhang et al., 1999). The primary conjugate of 12 genistein in both rats and humans is genistein-7-O-β-D-glucuronide, with minor conjugates being genistein-4’-O-β-D-glucuronide, genistein-4’-O-sulfate, genistein-7-O- sulfate, and diconjugate genistein-4’-O-sulfate-7-O-β-D-glucuronide (Zhou et al., 2008;
Bursztyka et al., 2008; Kwon et al., 2007). More genistein is converted into glucuronide conjugates following oral administration compared to after intravenous administration, likely due to significant first pass metabolism (Zhou et al., 2008).
Excretion: Rats excrete genistein primarily in bile, with a minor quantity excreted in urine (Zhou et al., 2008). In humans, more than 50% of genistein is excreted as genistein glucuronide via urine (Shelnutt et al., 2000). Mechanistically, this species difference is likely due to much more extensive enterohepatic recirculation in rats than in humans.
Bioavailability: Genistein glucuronide acts as a circulating store of genistein in the body since it can be deconjugated in the intestine by glucuronidase to release genistein. Enterohepatic recirculation is much more extensive in rats than it is in humans, thus contributing to greater bioavailability and prolonged pharmacological action of genistein in rats as compared to humans (Chen et al., 2003; Kwon et al., 2007). The poor bioavailability of genistein in humans is not due to poor absorption, but rather to its extensive metabolism and poor water solubility.
1.3.4. Environmentally relevant exposure levels
The average Japanese diet provides 0.21 to 0.43 milligrams of genistein per kilogram bodyweight, while typical Western diets contain 0.14 milligrams of genistein 13 per kilogram bodyweight. Circulating levels of genistein in human plasma are usually around 0.1 to 1 μM (Atherton et al., 2006), although levels can get as high as 25 μM in individuals following dietary supplementation with genistein (Atherton et al., 2006).
Infants fed soy-based formula consume between six and nine milligrams of genistein per kilogram bodyweight each day, resulting in circulating blood genistein concentrations of
5-10 μM (Xing et al., 2010).
14
1.4. Bisphenol A
Bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane; Figure 1.3.A] is one of the world’s most abundantly produced chemicals, with an annual increase in manufacturing demand of nearly 10% (Sala et al., 2010; Xing et al., 2010). BPA is a lipophilic synthetic industrial compound used in the synthesis of polycarbonate plastics and epoxy resins worldwide. The addition of BPA in polycarbonate plastics provides high mechanical strength, durability, and wear resistance, as well as good thermal stability, while maintaining high transparency (Sala et al., 2010; Brede et al., 2003). Its ubiquitous use in the synthesis of polycarbonate plastics and epoxy resins makes BPA a common additive in plastic baby bottles, food containers, and in the linings of food and beverage cans.
Thermal degradation and/or polymer degradation of polycarbonate products via heating and/or excessive wear can cause hydrolysis between ester bonds of BPA monomers, resulting in the liberation and migration of BPA from containers into contents, thus allowing for direct exposure to humans and animals via ingestion (Lim et al., 2009; Brede et al., 2003). Polycarbonate materials that are disposed of generally end up in either landfills or incinerators. Overtime, BPA can leach from landfills and escape into the environment, gaining entry into groundwater (Sala et al., 2010). Incinerating
BPA releases the compound as a vapor or soot, which also ultimately ends up in the environment. Although BPA has been removed from the formulations of many baby bottles in the Unites States and Canada, its worldwide use remains prevalent.
Furthermore, countless individuals continue to reuse plastic baby bottles that were originally synthesized with BPA. Additionally, BPA is commonly found in medical and 15 dental devices and sealants, wine vats, flame retardants, and thermal paper (Lyons, 2000;
Olea et al., 1996).
Human exposures to BPA are primarily through diet. Although BPA is not inherently found in food and beverage products, the containers that these products are stored and packaged in often contain BPA, which may easily leach from container into contents due to heating, excessive use, and/or alkaline conditions. Adult exposures to
BPA can reach as high as 59 µg/kg bw/day due to leaching from containers into food and beverage contents (Dekant and Volkel, 2008).
Endocrine disrupting properties of BPA have been reported by numerous laboratories across various species, both in vitro and in vivo (Xing et al., 2010; Kloas et al., 1999; vom Saal et al., 1998; Howdeshell et al., 1999). BPA is weakly estrogenic with greater affinity for ER-ß than for ER-α, with RBAs of 0.33 for ER-ß and 0.05 for
ER-α (Kuiper et al., 1997; Pritchett et al., 2002) (Table 1.1.). BPA’s predominant conjugate, BPA glucuronide, is unable to activate either ER-α or ER-ß (Matthews et al.,
2001). At concentrations as low as 10 μM, BPA has been shown to induce proliferation of MCF-7 human breast cancer cells in vitro (Perez et al., 1998). Exposure to environmentally relevant doses of BPA during gestation results in disrupted reproductive function in mice (Howdeshell et al., 1999). The induction of precocious puberty and disruption of postnatal growth rate are also results of exposure to environmentally relevant doses of BPA (Howdeshell et al., 1999). BPA has also been shown to inhibit embryonic development (Xing et al., 2010) and increase the incidence of hematopoeitic cancers (NTP, 2008). Upon extensive review of the compound’s deleterious biological effects across various species, the National Toxicology Program has declared that there is 16 concern that fetuses, infants, and children exposed to BPA will experience adverse effects to development and reproduction (NTP, 2008b).
17
1.5. Rationale for Analysis of Genistein and BPA
1.5.1. Comparable metabolism
Glucuronidation is the primary method of detoxification of both genistein and
BPA. The predominant metabolite of genistein and BPA in both rats and humans is its corresponding glucuronide (Elsby et al., 2001; NTP, 2007; Zhou et al., 2008; Bursztyka et al., 2008a; Kwon et al., 2007). The UGT isoforms that are primarily responsible for the glucuronidation of each genistein and BPA have been identified. The primary human
UGT isoforms implicated in the glucuronidation of genistein are the hepatic UGTs 1A1,
1A6, and 1A9, as well as extra-hepatic UGT1A10 which is present in the colon, but not in the liver (Doerge et al., 1999; Pritchett et al., 2008; Tang et al., 2009; Liu et al., 2007).
As previously discussed, UGT2B15 is the primary glucuronosyltransferase responsible for the glucuronidation of BPA in humans (Hanioka et al., 2008), while UGT2B1 is the primary isoform involved in the glucuronidation of BPA in rats (Yokota et al., 1999).
Previous studies have suggested that BPA inhibits the activity of UGT 1A6 via mixed competitive and non-competitive inhibition (Hanioka et al., 2008b), which could have implications in altered metabolism of genistein during binary exposures if UGT1A6 is a predominant isoform involved in the glucuronidation of genistein. Because UGTs aid in the detoxification of many endogenous and exogenous compounds, these enzymes serve as key players in the regulation of xenobiotic metabolism and toxicity, and their specific expression and activity can be altered by genetic and environmental factors, as well as by diet and exposure to different xenobiotics (Tukey and Strassburg, 2000). To be able to accurately assess the risk exerted by endocrine disrupting compounds, it is vital to 18 understand metabolic interactions between such compounds. A large focus is given to drug-drug interactions in regard to phase I metabolism by cytochrome p450 enzymes, but since glucuronidation is an effective detoxification pathway for most xenobiotics, the interactions between UGTs must be given attention as well.
1.5.2. Similar biological effects
As previously discussed, both genistein and BPA are estrogenic in nature and have been shown to exert endocrine disrupting effects across various species both in vitro and in vivo (Kuiper et al., 1997; Wisniewski et al., 2005; Ball et al., 2010; Xing et al.,
2010; vom Saal et al., 1998). Genistein and BPA both independently alter the proliferation of the human breast cancer cell line MCF-7 in vitro (Peterson et al., 1998;
Perez et al., 1998), and induce precocious puberty in vivo (Casanova et al., 1999;
Howdeshell et al., 1999). The two compounds share numerous common adverse biological endpoints, including inhibited embryonic development (Xing et al., 2010), altered postnatal growth rate (Ball et al., 2010), impaired reproductive function (Klein et al., 2002; Howdeshell et al., 1999), and altered sexually dimorphic development and behaviors (Wisniewski et al., 2005; Ball et al., 2010; Adwale et al., 2010), as discussed in sections 1.3.2. and 1.4.. Finally, both genistein and BPA exert pronounced adverse
effects when administered during prenatal and neonatal development as compared to
adulthood (Chen et al., 2007; Jefferson et al., 2005; Jefferson et al., 2006; Rubin et al.,
2001).
19
1.5.3. Common exposures
Not only do genistein and BPA share common routes of metabolism and exert similar adverse biological effects, but there are also abundant opportunities for simultaneous exposures, allowing for everyday co-exposures. Laboratory animals receiving soy diets and living in polycarbonate plastic cages are simultaneously exposed to genistein and BPA (Thigpen et al., 2004). A woman taking a menopausal relief aid containing genistein who recently had dental work performed that exposed her to BPA also experiences simultaneous exposure to the two endocrine disrupting compounds
(Evans et al., 2010; Howes et al., 2006; Olea et al., 1996). Other co-exposure scenarios include a man dining on a cheeseburger made with soy cheese eaten poolside from a polycarbonate plastic plate (Lim et al., 2009; Patisaul and Jefferson, 2010) and a hungry teenager enjoying homemade chili that contains soybeans and canned tomatoes (Evans et al., 2010; Olea et al., 1996). Of potentially highest concern, newborn babies, who are particularly vulnerable to the effects of endocrine disrupting compounds, are routinely exposed to both genistein and BPA simultaneously when fed a warm soy-based infant formula from a BPA-laden polycarbonate baby bottle (Sala et al., 2010; Brede et al.,
2003; Bhatia and Greer, et al., 2008) (Figure 1.4.).
Due to the ubiquitous nature of these compounds and abundant co-exposure scenarios, as well as comparable metabolism and similar biological effects, metabolic interactions between the two endocrine disrupting compounds are of particular interest.
Changes in with regard to rate of glucuronidation have been investigated in both human and rat liver microsomes in the presence of genistein and BPA.
20
2.0. AIM ONE: DETERMINATION VIA SPE AND HPLC-MS/MS
2.1. Abstract
Bisphenol A is a synthetic industrial reactant used in the production of polycarbonate plastics, and genistein is a natural phytoestrogen abundant in the soybean.
Current studies investigating the endocrine disrupting effects of concomitant exposures to
BPA and genistein have revealed the need for an analytical method for the simultaneous measurement of BPA and genistein, as well as their primary metabolites, bisphenol A ß-
D-glucuronide (BPA gluc) and genistein 4’-ß-D-glucuronide (genistein gluc), respectively. All four analytes were extracted from rat plasma via solid phase extraction
(SPE). Three SPE cartridges and four elution schemes were tested. Plasma extraction using Bond Elut Plexa cartridges with sequential addition of ethyl acetate, methanol, and acetonitrile yielded optimal average recoveries of 98.1% ± 1.8 BPA, 94.9% ± 8.0 genistein, 91.4% ± 6.1 BPA gluc, and 103% ± 6.1 genistein gluc. Identification and quantification of the four analytes were performed by a validated high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method using electrospray ionization and selective reaction monitoring. This novel analytical method should be applicable to the measurement of BPA, genistein, BPA gluc, and genistein gluc in urine, cultures, and tissue following in vivo exposures. While reports of the determination of
BPA and genistein independently exist, the simultaneous optimized extraction and detection of BPA, genistein, BPA gluc, and genistein gluc have not previously been reported prior to the work presented here. 21
2.2. Introduction
Endocrine disrupting compounds are chemicals that perturb the endocrine system either by acting as agonists or antagonists to sex hormone receptors, thus mimicking or preventing the action of endogenous estrogens and androgens. Laboratories have reported estrogenic activity for EDCs of various classes such as naturally occurring phytoestrogens (e.g., genistein, daidzein, biochanin A) (Casanova et al, 1999; Peterson and Barnes, 1993), man-made plasticizers (e.g., bisphenol A, phthalates) (Richter et al,
2007; Rubin et al, 2001; Hannas et al, 2011), pesticides (e.g., methoxychlor, atrazine)
(Armenti et al, 2008), and other synthetic industrial chemicals (e.g., polybrominated diphenyl alkanes) (Perez et al, 1998).
BPA [2,2-bis(4-hydroxyphenyl)propane] (Figure 1.3.A) is a monomer widely employed in the synthesis of polycarbonate plastics and epoxy resins worldwide, and is commonly found in baby bottles, food containers, and linings of food and beverage cans.
BPA can migrate from containers into contents, allowing for direct exposure to humans via ingestion (Lim et al, 2009). BPA is weakly estrogenic (Kuiper et al, 1998), and numerous laboratories have demonstrated endocrine disrupting properties of BPA including in vitro proliferation of MCF-7 human breast cancer cells (Perez et al, 1998) and in vivo alteration of reproductive function and postnatal growth rate (Xing et al,
2010; vom Saal et al, 1998; Howdeshell et al, 1999). After much review of the risk of human exposure to BPA, the National Toxicology Program declared that there is some concern for adverse effects to human development and reproduction, specifically on the behavior, brain, and prostate gland of fetuses, infants, and children exposed to BPA
(NTP-CERHR, 2008). 22
Genistein [4’,5,7-trihydroxyisoflavone] (Figure 1.1.A) is a naturally occurring phytoestrogen found in many legumes and grains, with soybeans being a particularly rich source. Since nearly 60% of all packaged foods contain soy, humans consume a nearly constant supply of genistein in their everyday diets (Patisaul and Jefferson, 2010). The majority of commercially available laboratory rodent diets naturally contain variable concentrations of genistein, usually in the form of soybean meal or soy protein, thus making genistein a ubiquitous component of laboratory rodent chow (Thigpen et al,
2004). Like BPA, genistein exhibits weak estrogenic activity both in vitro and in vivo
(Kuiper et al, 1998; Shelnutt et al, 2000). The interpretation of data on the effects of genistein is controversial, with some groups focusing on the beneficial effects of genistein such as the phytoestrogen’s chemotherapeutic properties and ability to prevent osteoporosis (Albertazzi, 2002; Akiyama et al, 1987), while other laboratories emphasize genistein’s ability to cause adverse health effects in humans and laboratory animals, particularly endocrine disruption (Messina et al, 2006; Ball et al, 2010). Genistein has been shown to be detrimental to the differentiation and development of reproductive organs in embryos, fetuses, and neonates, suggesting that there is a critical window of exposure which may lead to latent effects (Patisaul and Polston, 2008; Jefferson et al,
2002).
Analyses of BPA and genistein have increased in recent years due to their emergence as prevalent endocrine disrupting compounds. Methods have been previously established for the extraction and detection of either BPA or genistein. Established methods allow for the extraction of BPA from environmental water samples, as well as from biological fluids including plasma, serum, amniotic fluid, breast milk, and urine (Yi 23 et al, 2010; Rezaee et al, 2009). Genistein has been routinely extracted from food and beverage products, as well as urine, serum, and plasma (Valentin-Blasini et al, 2000;
Twaddle et al, 2002; Wilkinson et al, 2002). Solid phase extraction is the predominant isolation method for both BPA and genistein. Traditional SPE sorbent materials such as
C18 and other reversed phase sorbents are commonly used to extract BPA (Xiao et al,
2006), while more unique SPE sorbents such as bamboo-activated charcoal are reportedly utilized as well (Zhao et al, 2010). Current leading methods for the extraction of genistein employ C18 or Oasis HLB SPE cartridges (Valentin-Blasini et al, 2000).
Methods for separation and detection of BPA or genistein include HPLC-ESI/MS/MS
(Zhao et al, 2010), HPLC-APCI/MS/MS (Valentin-Blasini et al, 2000), HPLC-UV
(Hosoda et al, 2008), and UPLC-ESI/MS/MS (Churchwell et al, 2005).
Several studies have maximized the recovery and/or detection of either BPA or genistein, but no studies to date have focused on optimizing the simultaneous measurement of the two compounds. BPA and genistein are rarely analyzed together, possibly due to the fact that analytes of interest and methodologies are often grouped on the basis of chemical structure similarities or are members within the same compound class, such as pesticides or phytoestrogens. The prevalence of co-exposures to BPA and genistein clearly warrant their simultaneous analysis. There are a number of scenarios in which individuals may be exposed to BPA and genistein simultaneously, such an infant drinking soy-based formula from a polycarbonate baby bottle (Figure 1.4.), or a woman taking a menopausal relief aid containing genistein who recently had dental work performed that exposed her to BPA (Howes et al, 2006, Olea et al, 1996). In this work,
BPA and genistein are analyzed simultaneously because they are relevant in common 24 binary exposure scenarios, in addition to the fact that they share numerous common biological endpoints, including precocious puberty, irregular cyclicity, and altered cellular differentiation (Casanova et al, 1999; Rubin et al, 2001; Howdeshell et al, 1999).
Synergistic effects of inhibited embryonic development in rat embryos to concomitant exposures of genistein and BPA have been reported, suggesting that altered metabolism may contribute to such an observed biological effect (Xing et al., 2010).
The ability to simultaneously recover and detect BPA and genistein and their primary metabolites is immensely beneficial because it requires smaller sample sizes, decreased costs of materials and personnel, and reduced analysis time. The work presented here is significant because it provides a novel analytical method for the simultaneous measurement of BPA, genistein, BPA gluc, and genistein gluc.
25
2.3. Materials and Methods
BPA, genistein, and ammonium acetate were purchased from Sigma-Aldrich (St.
Louis, MO). BPA gluc and genistein gluc were purchased from TRC Canada (North
York, Ontario). Ethyl acetate (pesticide grade), methanol (for LC/MS), and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ). Honeywell Burdick and Jackson
(Muskegon, MI) supplied high purity solvents acetonitrile and water, both of high performance liquid chromatography (HPLC) grade.
The extraction of BPA, BPA gluc, genistein, and genistein gluc from rat plasma was performed via SPE. Three cartridges were tested for efficiency of co-extraction:
Bond Elut Plexa cartridges (Varian Inc., Palo Alto, CA; 60 mg, 1 mL), Oasis
Hydrophilic-Lipophilic Balance (HLB) cartridges (Waters, Milford, MA; 30 mg, 1 mL), and United Chemical Technologies (UCT) C18 cartridges (United Chemical
Technologies, Bristol, PA; 100 mg, 1 mL). Bond Elut Plexa cartridges feature hydrophilic surfaces and a gradient of polarity that allow for good transfer of small analytes into the polymer core, while excluding large proteins, and generally show efficient extraction of analytes across a broad range of polarities and acid/base properties.
Oasis HLB cartridges are routinely used to extract parent compounds as well as their polar metabolites. UCT C18 cartridges are hydrophobic, featuring a sorbent that is composed of a silica backbone with hydrocarbon chains; these relatively non-selective cartridges are often used to extract non-polar or neutral analytes from complex matrices.
Various combinations of ethyl acetate, methanol, and acetonitrile were used as elution solvents, with each combination tested per cartridge type.
26
2.3.1. Solid phase extraction
All samples had a total volume of 1.0 mL and consisted of 100 µL of rat plasma with citrate (Fisher Scientific, Pittsburgh, PA), 100 µL of 250 mM ammonium acetate
(pH 5), 80 µL of 1M formic acid, water, and 0.1 µg/mL BPA, BPA gluc, genistein, and genistein gluc. Samples were sonicated for five minutes then centrifuged at 2500 revolutions per minute for ten minutes at 4˚C. Each SPE cartridge was conditioned with three mL of methanol followed by three mL of water before samples were loaded. Two mL of 9:1 water/methanol (v/v) wash solution was passed through each column prior to elution. The optimized elution was performed at a flow rate of 1-2 mL/min with one of the following elution schemes, with elution solvents added sequentially in the order they are listed: three mL ethyl acetate and three mL methanol (EM), three mL ethyl acetate and three mL acetonitrile (EA), three mL methanol and three mL acetonitrile (MA), or three mL ethyl acetate, two and a half mL methanol, and two and a half mL acetonitrile
(EMA). SPE extracts were evaporated to dryness under vacuum and a stream of nitrogen then reconstituted in 200 μL of 75% acetonitrile in water.
2.3.2. Standard solutions
Initial genistein stock solutions were prepared by dissolving genistein in methanol, whereas BPA, BPA gluc, and genistein gluc were initially dissolved in acetonitrile. Subsequent standards for all analytes were prepared via serial dilution in acetonitrile. Standard solutions ranged from 1 µg/mL to 1000 µg/mL, and were stored at
-20ºC. 27
2.3.3. Blank controls
Blank controls contained high purity water in place of standard solutions and were processed alongside samples, using all of the same supplies and reagents. While blank controls are always important, they are especially vital when analyzing ubiquitous compounds such as BPA, which is known to be prevalent in many common laboratory supplies and other materials required for SPE and HPLC-MS/MS analysis due to its use as a plasticizer (Stiles et al, 2007). Efforts were made to minimize the contact of samples with plastics, and plasticware was replaced by glassware wherever possible. Despite significant efforts made to minimize the use of plastic materials by substitution with glass products, contamination with BPA is still a common challenge in the laboratory. Blank controls were performed for each cartridge type and elution scheme.
2.3.4. Chromatographic conditions
10 μL of each reconstituted extract was directly injected via a Finnigan Surveyor
Autosampler Plus (Thermo Fisher Scientific, Waltham, MA). Chromatographic separation was carried out with either a Kinetex C18 column (Phenomenex, Torrance,
CA; 100 x 4.6 mm I. D., 2.6 µm) or a Discovery C8 column (Supelco, St. Louis, MO; 50 x 4.6 mm I.D., 5 μm). A Krud Katcher Ultra In-Line Filter guard column (Phenomenex;
0.5 µm) was used with both HPLC columns. Mobile phase A was 2 mM ammonium acetate (pH 9), and mobile phase B was acetonitrile. Replacing acetonitrile with methanol in the mobile phase solution has been known to contribute to poor peak shape and variable baseline (Xiao et al, 2006). For this reason, we chose to use acetonitrile 28 rather than methanol in our mobile phase. Optimal chromatographic separation relative to discrete non-overlapping peaks with distinct baseline resolution between xenoestrogen analytes of interest and interfering substances was achieved (Figure 2.1.A, Figure 2.1.B,
Figure 2.2.A, Figure 2.2.B) when the following gradient was employed: 0 to 3 min 50%
B, 3 min to 14 min 50% to 90% B, 14 min to 18 min 90% B, 18 min to 18.2 min 90% to
50% B, 18.2 min to 20 min 50% B, using a Finnigan Surveyor MS Pump Plus (Thermo
Fisher Scientific). Flow rate was maintained at 250 µL/min for the duration of each twenty minute analysis.
2.3.5. Mass spectrometry
The HPLC eluate was directed into a Thermo LTQ mass spectrometer (Thermo
Fisher Scientific), using electrospray ionization (ESI) in negative ion mode, and a linear ion trap as an analyzer, all under the control of Xcalibur 2.0.7 software. Optimized ESI source conditions include a sheath gas flow rate of 40 (arbitrary units), spray voltage of
5.0 kV, heated capillary temperature of 275°C, capillary voltage of -25 V, and spray current of 3.0 μA. To achieve optimal sensitivity, parameters including tube lens offset, multipole offset, gate and front lens voltages, and multipole RF amplitude were optimized prior to each analysis using a 1 μg/mL standard solution of BPA that was infused at a flow rate of 3 μL/min together with HPLC mobile phase at a flow rate of 250
μL/min. Data was acquired in the selective reaction monitoring (SRM) mode with an isolation width of a mass-to-charge ratio (m/z) of 2 for each analyte. Product ions were generated by applying a collision energy of 35 for BPA, BPA gluc, and genistein gluc, 29 and a collision energy of 40 for genistein. Mass spectrometric parameters are summarized in Table 2.1. High purity nitrogen was used as the sheath gas.
2.3.6. Statistical analyses
Data are reported as average recovery ± relative standard deviation (RSD).
Single-point calibrations to serve as quality control were prepared by spiking known concentrations of each analyte into mobile phase, and were directly injected into the
HPLC-MS/MS system multiple times. An average of 10 or 11 replicate injections of quality control standard was calculated. The blank-subtracted quantity of each analyte measured in the corresponding unknown samples was then compared to the mean quantity of analyte detected in replicate injections of spiked standard. This method of using a single-point calibration was used to verify that instrumental drift was not a major factor, since analytical runs often exceed 40 hours of continuous HPLC-MS/MS analysis.
Reported values have been blank-subtracted from raw data prior to calculation of percent recovery. Three separate extractions were performed per cartridge type and elution scheme, and each reconstituted extract was injected into the HPLC-MS/MS three times.
Data acquisition was performed using Xcalibur 2.0.7, and data were analyzed by Qual
Browser 2.0.7 (Thermo Fisher Scientific). Quantitation was performed with Excel 2003, based on manually integrated peak areas using genesis peak integration and 15-point smoothing with Xcalibur 2.0.7. Residual error values were calculated using Stata 11.2
(Stata Corp., College Station TX).
30
2.4. Results
2.4.1. Retention times, limits of detection, and calibration curves
Retention times with the Kinetex C18 column were approximately 6.7, 2.7, 3.5, and 2.7 minutes for BPA, BPA gluc, genistein, and genistein gluc, respectively (Figure
2.1.A and Figure 2.1.B). When separated with the Discovery C8 column, retention times for BPA, BPA gluc, genistein, and genistein gluc were approximately 4.0, 1.6, 2.5, and
1.7 minutes, respectively (Figure 2.2.A, Figure 2.2.B, Table 2.2.). On-column limits of detection were 100, 250, 50, and 25 picograms for BPA, BPA gluc, genistein, and genistein gluc, respectively. To quantitate the amount of each analyte, separate calibration curves were prepared over the ranges of 0.1 μg/mL and 5 μg/mL for all four analytes. Standard solutions were injected into the HPLC-MS/MS system, and calibration curves were obtained using integrated area. All analytes of interest showed good linearity between 0.1– 5 μg/mL, with r2 values of 0.999, 0.999, 0.995, and 0.997 for
BPA, BPA gluc, genistein, and genistein gluc, respectively. Residual error values ranged
from 0.08% to 2.37%.
2.4.2. Recoveries
Recoveries of BPA (Figure 2.3.A) and genistein (Figure 2.3.B) varied among
cartridge types as well as elution scheme. Overall background subtracted recoveries
ranged from 62.1% to 108% for BPA and 4.28% to 108% for genistein.
31
2.4.2.1. Bond Elut Plexa SPE cartridges
All four elution schemes resulted in adequate recoveries for both BPA and genistein. EMA recovered the greatest amount of BPA, followed by EM, EA, and MA
(98.1% ± 1.8, 96.4% ± 18, 93.9% ± 11, and 78.6% ± 16, respectively). Elution with
EMA also yielded substantially more reproducible results than any other elution scheme, as expressed by RSD. The recoveries of genistein by the different elution solvents, in order from greatest recovery to least were: 108% ± 20 using EM, 108% ± 11 using EA,
94.9% ± 8.0 using EMA, and 92.9% ± 8.2 using MA.
2.4.2.2. Oasis HLB SPE cartridges
When analyzed across elution scheme, EMA yielded the greatest recovery of BPA on Oasis HLB cartridges, followed by EA, EM, and MA, with recoveries of 108% ± 16,
85.5% ± 12, 67.2% ± 14, and 62.1% ± 9.9, respectively. Genistein recoveries from SPE with Oasis HLB cartridges were also greatest when eluted with EMA, followed by MA,
EM, and EA, 90.4% ± 9.7, 83.9% ± 7.2, 83.7% ± 14, and 4.28% ± 21, respectively.
2.4.2.3. UCT C18 SPE cartridges
As observed with both of the aforementioned cartridges, BPA recovery was maximized when eluted with EMA from UCT C18 cartridges. The average recovery was
107% ± 10 with EMA, followed by 87.0% ± 10 with EM, 77.1% ± 11 with EA, and
70.6% ± 12 with MA. Genistein recoveries from SPE with UCT C18 cartridges were 32
96.7% ± 15 using MA, 92.1% ± 4.1 using EMA, 90.7% ± 14 using EA, and 82.3% ± 7.1 using EM.
2.4.2.4. Glucuronide recoveries
The analytical method presented here focuses on the optimization of the simultaneous measurement of BPA and genistein. Upon optimization, the method was tested for its ability to recover and detect the primary glucuronides of BPA and genistein, which are each more hydrophilic than their parent compound (Figure 1.1. and Figure
1.3.). Despite these differences in water solubility, the glucuronides were successfully eluted with the optimized method, using Bond Elut Plexa cartridges and elution with
EMA. Elution of the glucuronides using the optimized SPE conditions yielded background-subtracted average recoveries of 91.4% ± 6.1 for BPA gluc and 103% ± 6.1 genistein gluc (Figure 2.4.), demonstrating that the novel method presented here is effective at recovering and detecting not only BPA and genistein, but also their primary metabolites. Overall background subtracted recoveries for BPA gluc and genistein gluc ranged from 85.9% to 101% and 91.4% to 110%, respectively.
2.4.3. HPLC column performance
For both columns, discrete separation of BPA and genistein with distinct baseline resolution was obtained within seven minutes. On both columns, BPA gluc was the first analyte to elute, followed by genistein gluc, genistein, and finally BPA. On average, all 33 four analytes eluted within a range of four and a half minutes on the Kinetex C18 column and within two and a half minutes on the Discovery C8 column (Table 2.2.).
Reproducibility of replicate injections performed with the Discovery C8 column was far superior to that of the Kinetex C18 column (RSDs 3.6 and 13 for six replicate BPA injections on Discovery C8 and Kinetex C18, respectively). The Kinetex C18 column was stable over a pH range of 1.5 to 10, while the Discovery C8 column was stable between pH 2 to 7.5. With the solvent gradient used in this method, the measured pH ranged from 5 to 7, which fell within the stability range for both columns.
34
2.5. Discussion
Elution with EMA resulted in the greatest recovery of BPA for all three cartridge types (Figure 2.3.A). SPE with Bond Elut Plexa cartridges yielded greatest recovery of
BPA for all elution schemes except for elution with EMA (98.1% ± 1.8), which was not appreciably less than that observed with either Oasis HLB (108% ± 16) or UCT C18
(107% ± 10). Additionally, the variability of BPA recovery among SPE replicates eluted from Bond Elut Plexa cartridges with EMA is substantially less than that observed with
Oasis HLB or UCT C18 cartridges (RSDs 1.8, 16, and 10, respectively). Collectively, these data indicate that elution with EMA from Bond Elut Plexa cartridges provides optimal recovery of BPA.
Elution of genistein via SPE with Bond Elut Plexa cartridges yielded greatest genistein recovery for all four elution schemes, compared to the other cartridges tested.
The exception was MA, where the average recovery of genistein from Bond Elut Plexa was very near the recoveries from both other cartridge types (Figure 2.3.B). While EMA did not yield the overall greatest recovery of genistein from Bond Elut Plexa cartridges, its recovery was not substantially less than that observed with the other elution schemes.
While some mean recoveries exceed 100%, none fall outside the acceptable range for measurement error of ± RSD of 20. When extracted with Bond Elut Plexa cartridges, elution with EMA resulted in markedly less variability among replicate samples, as compared to the other elution schemes.
Reproducibility is an important factor when determining ideal extraction conditions. When considering reproducibility in conjunction with the recovery efficiency for both BPA and genistein, it is clear that optimal simultaneous recoveries for BPA and 35 genistein are achieved by SPE performed on the Bond Elut Plexa cartridge, with sequential additions of three mL ethyl acetate, two and half mL methanol, and two and half mL acetonitrile. This optimized method also demonstrated superb recovery, efficiency, and reproducibility when used to extract the primary metabolites of BPA and genistein from rat plasma, achieving average recoveries of 91.4% ± 6.1 and 103% ± 6.1 for BPA gluc and genistein gluc, respectively (Figure 2.4.).
The on-column detection limits reported here are not as low as those achieved using a triple quadrupole mass spectrometer as an analyzer (Valentin-Blasini et al, 2000).
Additionally, analytical methodologies aimed to measure individual analytes are often capable of achieving lower limits of detection than those presented here; however in the present method, it was acceptable to compromise higher detection limits for a comprehensive method allowing for the simultaneous measurement of BPA, BPA gluc, genistein, and genistein gluc.
The Kinetex C18 column utilizes a fused-core silica particle technology that is credited with increasing resolution, throughput, and sensitivity for Ultra-High
Performance Liquid Chromatography analyses; unfortunately, it proved disadvantageous in the analytical method presented here. When compared to the Discovery C8 column for use in the present method, it became apparent that the Kinetex C18 column was inferior.
The Discovery C8 HPLC column displayed dramatically increased reproducibility among replicate injections compared to the Kinetex C18 HPLC column. Additionally, variability of results when using the Kinetex C18 column increased substantially after only 200 total injections, whereas variability remained minimal after over more than
1000 injections on the Discovery C8 column, making the longevity of Discovery C8 36 dramatically better than Kinetex C18. Due to superior column lifetime and reproducibility, Discovery C8 was selected as the HPLC column of choice for detection of BPA, genistein, BPA gluc, and genistein gluc.
37
2.6. Conclusion
In this work, a highly sensitive SPE isolation method was coupled with LC-ESI-
MS/MS quantitation for the simultaneous measurement of endocrine disrupting compounds BPA, genistein, BPA gluc, and genistein gluc. Bond Elut Plexa SPE cartridges with ethyl acetate, methanol, and acetonitrile elution produced optimal recoveries of 98.1% ± 1.8 for BPA, 91.4% ± 6.1 for BPA gluc, 94.9% ± 8.0 for genistein, and 103% ± 6.1 for genistein gluc and were more reproducible than either the Oasis or
UCT SPE cartridges tested. This analytical method allows for BPA, BPA gluc, genistein, and genistein gluc to be efficiently recovered from plasma in a single comprehensive method, allowing for the analysis of in vitro and in vivo toxicology exposure studies where BPA, genistein, and their primary metabolites are present. This analytical method may be adapted for the measurement of BPA, genistein, and their glucuronides from other biological matrices including urine, cultures, and possibly tissue, following exposures.
Portions of Section 2.0. have been published in Analytical and Bioanalytical Chemistry:
Coughlin JL, Winnik B, and Buckley B. (2011). Measurement of bisphenol A, bisphenol A β-D-glucuronide, genistein, and genistein 4'-β-D-glucuronide via SPE and HPLC-MS/MS. Analytical and Bioanalytical Chemistry. 401, 995- 1002.
38
3.0. AIM TWO: INHIBITION OF GLUCURONIDATION
3.1. Abstract
Genistein is a natural phytoestrogen abundant in the soybean, and BPA is a synthetic chemical used in the production of polycarbonate plastics and epoxy resins.
Both genistein and BPA are weakly estrogenic and disrupt the endocrine system both in vivo and in vitro. Growing concerns of altered xenobiotic metabolism due to concomitant exposures of the two endocrine disruptors from soy milk in BPA-laden baby bottles
(Figure 1.4.) has warranted in vitro analysis of the rate of glucuronidation of genistein in the absence (0 μM) and presence (25 μM) of BPA by mixed gender human liver microsomes (HLMs) and rat liver microsomes (RLMs). HLMs yield a maximal reaction rate of genistein glucuronidation (Vmax) of 0.93 ± 0.10 nmol/min/mg protein in the
absence of BPA and 0.62 ± 0.05 nmol/min/mg protein in the presence of BPA. Km values for the glucuronidation of genistein by HLMs in the absence and presence of BPA are 15.1 ± 7.9 μM and 21.5 ± 7.7 μM, respectively, resulting in a Ki of 58.7 μM. A significantly reduced Vmax and unchanged Km when co-incubated with 25 μM BPA
indicates that BPA non-competitively inhibits the glucuronidation of genistein in human liver microsomes. In rat liver microsomes, the addition of 25 μM BPA resulted in a
negligible change in Vmax (2.91 ± 0.26 nmol/min/mg in absence of BPA and 3.05 ± 0.41
nmol/min/mg in the presence of BPA) and an increase in apparent Km (49.4 ± 14 μM in
the absence of BPA and 84.0 ± 28 μM in the presence of BPA), indicative of competitive
inhibition. These findings are significant because they not only suggest that BPA is 39 capable of inhibiting the glucuronidation of genistein in vitro, but also that the type of inhibition is different between human liver microsomes and rat liver microsomes with implications for the application of animal models to assess human risk.
40
3.2. Introduction
Glucuronidation, a major form of phase II xenobiotic metabolism, is catalyzed by various isoforms belonging to the UDP-glucuronosyltransferase (UGT) enzyme family.
UGTs are promiscuous enzymes, capable of conjugating various structurally diverse substrates. In general, UGTs attack their substrates at nucleophilic sites containing oxygen (e.g., hydroxyls, carboxylic acids), nitrogen (e.g., amines), sulfur (e.g., thiols), and occasionally carbon. UGTs are constitutively expressed in a tissue-specific manner, with their expression and activity altered by genetic and environmental factors (Tukey and Strassburg, 2000). While a few UGTs are known to exist only in extra-hepatic tissues, most UGTs are expressed abundantly in the liver, specifically within microsomes, the subcellular fraction of hepatocytes that are rich in xenobiotic metabolizing enzymes
(Tukey and Strassburg, 2000; Miners et al, 2006). Although UGT isoforms vary between species, several inter-species homologues have been identified. Humans and rats have several orthologous UGTs, including 1A1, 1A3, 1A5, 1A6, 1A7, 1A8, and 1A10, while humans, but not rats, also have functional UGTs 1A4 and 1A9 (Mackenzie et al, 2005).
In general, rats have an overall greater capacity for glucuronidation compared to humans
(Elsby et al, 2001; Volkel et al, 2002).
The UGT active site is located along the luminal membrane of the smooth endoplasmic reticulum. In order for the UGT active site to come in contact with its substrate in vitro, pore-forming agents such as alamethicin must be used to perforate the membrane of the endoplasmic reticulum (Kilford et al, 2009). Along with the required
UDPGA, UGTs catalyze the synthesis of a β-D-glucuronide conjugate. Typically, glucuronide conjugates are less biologically active and more readily available for 41 excretion than their parent substrates, thus making UGTs key players in the regulation of xenobiotic metabolism and toxicity.
Genistein [4’,5,7-trihydroxyisoflavone, Figure 1.1.] is a natural phytoestrogen present in abundant quantities in the soybean. Since nearly sixty percent of all processed foods in today’s market contain soy, often in the form of textured soy protein or soy protein isolate, humans consume a nearly continuous supply of genistein in their everyday diet (Patisaul and Jefferson, 2010). The conflicting effects of genistein remain controversial. While some laboratories report the therapeutic effects of genistein such as chemoprevention, improved bone health, and amelioration of menopausal symptoms
(Goldwyn et al, 2000; Evans et al, 2010), other groups emphasize the phytoestrogen’s adverse health effects, specifically endocrine disruption (Jefferson et al, 2005; Casanova et al, 1999). Genistein has been shown to inhibit embryonic development, disrupt reproductive development, alter cellular differentiation and postnatal growth rate, decrease aggressive behaviors, and impair spatial learning (Ball et al, 2010; Xing et al,
2010; Wisniewski et al, 2005).
BPA [4,4’-isopropylidenediphenol, Figure 1.3.] is a synthetic monomer used in the synthesis of polycarbonate plastics and epoxy resins. BPA is a fairly ubiquitous compound, found as a common component in plastic baby bottles, as well as food and beverage containers. Because BPA can leach from plastic containers into contents, especially when containers are exposed to heat and/or excessive wear, humans are at a direct risk of exposure (Brede et al, 2003). BPA has been shown in various laboratories to have estrogenic activity across numerous species both in vitro (100 μM) and in vivo (2
μg/kg bw/day) (Kuiper et al, 1998; Perez et al, 1998; vom Saal et al, 1998). Endocrine 42 disrupting properties of BPA are routinely reported, and include inhibited embryonic development (Xing et al, 2010), altered postnatal growth rate, and induction of precocious puberty (Howdeshell et al, 1999).
The UGT isoforms that are primarily responsible for the glucuronidation of genistein and BPA have been identified. The primary human UGT isoforms implicated in the glucuronidation of genistein are hepatic UGTs 1A1, 1A6, and 1A9, as well as extra-hepatic UGTs 1A10 and 2B17 (Doerge et al, 1999; Pritchett et al, 2008; Tang et al,
2009; Liu et al, 2007). Although the predominant UGTs responsible for the glucuronidation of genistein in rats have not been definitely identified, they are suspected to be UGTs 1A1, 1A6, and 1A10, since these isoforms are orthologous to the human
UGTs (Tukey and Strassburg, 2000). The predominant UGT responsible for the glucuronidation of bisphenol A in rats is UGT2B1, which is homologous to human UGTs
2B7 and 2B17, neither of which is the primary UGT involved in BPA glucuronidation in humans (Elsby et al, 2001). Instead, human UGT2B15 is the primary glucuronosyltransferase responsible for the glucuronidation of BPA in humans (Hanioka et al, 2008).
The goal of the present study was to investigate the inhibitory effect of BPA on the glucuronidation of genistein in vitro during simultaneous exposure to both endocrine disrupting compounds, simulating a concomitant exposure. Hepatic UGT inhibition was investigated in both human and liver microsomes using binary incubations of BPA and genistein.
43
3.3. Materials and Methods
Chemicals were purchased from the sources indicated: Bisphenol A, genistein, ammonium acetate, UDPGA, alamethicin, and magnesium chloride (Sigma-Aldrich, St.
Louis, MO); BPA gluc and genistein gluc (TRC Canada, North York, Ontario); ethyl acetate (pesticide grade), methanol (LC/MS grade), and formic acid (Fisher Scientific,
Fair Lawn, NJ); and high purity solvents acetonitrile and water, both of HPLC grade
(Honeywell Burdick and Jackson, Muskegon, MI). Bond Elut Plexa cartridges (60 mg, 1 mL) were purchased from Varian Inc. (Palo Alto, CA). Human liver microsomes
(HLMs; pooled from 50 donors, mixed gender) and rat liver microsomes (RLMs; pooled from 100 female and 100 male Wistar rats) were purchased from XenoTech (Lenexa,
Kansas).
3.3.1. Glucuronide formation
Reagent pool was prepared by mixing together HLMs (0.0625 mg/mL) or RLMs
(0.025 mg/mL) with alamethicin (25 µg/mg of protein in incubation), magnesium chloride (4 mM), and Tris-HCl buffer (pH 7.4, 50 mM). Microsomal protein concentrations were optimized through range-finding experiments with varying concentrations of HLMs and RLMs ranging between 0.015 to 0.5 mg/mL (Figure 3.1. and Figure 3.2.). For enzyme kinetics studies, varying concentrations of either BPA or genistein were added (10, 25, 50, 100, 175, and 250 μM). Equal aliquots of reagent pool were placed in incubation vials (1.5 mL center drain glass vials, Sigma) and pre- incubated in a shaking water bath (80 strokes/min) at 37˚C for 15 minutes. Reactions 44 were initiated by the addition of UDPGA (5 mM), bringing each incubation volume to a total of 200 µL. After a 30 minute incubation period, reactions were terminated via addition of 400 µL of ice-cold acetonitrile. Duration of incubation was optimized by testing and analyzing total metabolite formation after 0, 15, 30, 45, and 60 minute incubations. Samples were then centrifuged at 2,800 rpm for 20 minutes and supernatant was collected for eventual SPE cleanup and HPLC-MS/MS analysis.
3.3.2. Inhibition studies
Reagent pools were prepared as previously described. To determine Ki, samples
contained varying concentrations of genistein (10, 25, 50, 100, 175, 250 μM) in the
absence (0 μM) and presence (25 μM) of BPA. For IC50 determination in HLMs,
samples contained varying concentrations of BPA (0, 5, 10, 25, 50, 100, 250 μM) in the
presence of 100 μM genistein.
3.3.3. Sample preparation
Analytes were isolated from their matrix using SPE with Bond Elut Plexa
cartridges and sequential additions of the elution solvents ethyl acetate, methanol, and
acetonitrile, as previously described by Coughlin et al, 2011. SPE extracts were
evaporated to dryness under vacuum and a stream of nitrogen before being reconstituted
in 100 μL of 50% acetonitrile in water. 45
3.3.4. Standard solutions
Initial genistein stock solutions were prepared by dissolving genistein in methanol; BPA, BPA gluc, and genistein gluc were initially dissolved in acetonitrile. All subsequent standards, ranging from 1 µg/mL to 1000 µg/mL, were prepared via serial dilution in acetonitrile and were stored at -20ºC.
3.3.5. Blank controls
Two sets of blank controls were used: incubation blanks and SPE blanks.
Incubation blank controls contained all of the same reagents and substrates as the samples, however, the blanks were terminated via addition of ice-cold acetonitrile prior to initiation of the reaction with UDPGA. SPE blank controls contained high purity water in place of standard solutions, and were processed alongside samples during SPE using all of the same supplies and reagents. The use of blank controls is especially important when analyzing ubiquitous compounds such as BPA, which is known to be prevalent in numerous laboratory supplies due to its use as a plasticizer (Stiles et al, 2007). The use of plastics was minimized wherever possible, such as using glass incubation vials instead of plastic, in an effort to reduce contamination with BPA.
3.3.6. Analytical conditions
Analytical conditions were similar to those optimized by Coughlin et al, 2011 and described in Chapter 2. Briefly, 10 μL of each reconstituted SPE extract was injected via 46 a Finnigan Surveyor Autosampler Plus (Thermo Fisher Scientific, Waltham, MA).
Chromatographic separation was carried out with a Discovery C8 column (Supelco, St.
Louis, MO; 50 x 4.6 mm I.D., 5 μm) and a Krud Katcher Ultra In-Line Filter guard column (Phenomenex; 0.5 µm). Mobile phase was delivered at a constant flow rate of
250 µL/min using a Finnigan Surveyor MS Pump Plus (Thermo Fisher Scientific). A gradient of solution A (10% acetonitrile in 2 mM ammonium acetate, pH 9), and solution
B (acetonitrile) was used as follows: 0 to 3 min 45% B, 3 min to 14 min 45% to 89% B,
14 min to 18 min 89% B, 18 min to 18.2 min 89% to 45% B, 18.2 min to 20 min 45% B.
The HPLC eluate was directed to a Thermo LTQ mass spectrometer (Thermo Fisher
Scientific), using negative ion mode electrospray ionization (ESI) and a linear ion trap, all regulated via Xcalibur 2.0.7 software. Data was acquired using selective reaction monitoring (m/z 227 for BPA, m/z 403 for BPA gluc, m/z 269 for genistein, and m/z 445 for genistein gluc] with an isolation width of 2 m/z for each analyte. Daughter ions (m/z:
212, 227, 181, and 269 for BPA, BPA gluc, genistein, and genistein gluc, respectively) were generated by applying a collision energy of 35 for BPA, BPA gluc, and genistein gluc, and a collision energy of 40 for genistein. High purity nitrogen was used as the sheath gas.
3.3.7. Data analyses
All incubations for enzyme activity and inhibition were performed in triplicate for each substrate concentrate, and each reconstituted extract was injected into the HPLC-
MS/MS in triplicate. Replicate injections were averaged together for each sample. All 47 incubations were performed three times, and reported values represent the overall average of these nine determinations ± RSD. Reported values have been blank-subtracted from raw data prior to calculation of analyte quantity. Michaelis-Menten values of apparent
Km and Vmax were obtained by analyzing Lineweaver-Burk plots using GraphPad Prism
5.04 (La Jolla, CA), and confirmed via manual calculation using the Michaelis-Menten equation:
(V max)[S] V0 = (Km [S])
where Vmax represents the maximal reaction rate, [S] represents the substrate concentration, and Km is the Michaelis constant, which is defined as the substrate
concentration at half of the maximal reaction rate (Golan et al., 2005). Ki values were
found graphically using Lineweaver-Burk plots, and verified via manual calculation
using the following equation:
[I] Km apparent = (1 + ) • Km Ki
where Km apparent is the Michaelis constant in the presence of inhibitor, Km is the Michaelis
constant in the absence of inhibitor, [I] represents the inhibitor concentration, and Ki
represents the dissociation constant. IC50 values were found graphically based on plots
created using GraphPad Prism 5.04. Unpaired student t-tests were performed using
GraphPad Prism 5.04 to determine statistical significance between curves of 0 and 25 μM
BPA. Data acquisition was performed using Xcalibur 2.0.7 and analyzed using Qual
Browser 2.0.7 (Thermo Fisher Scientific). Quantitation was calculated in Excel 2003, 48 using manually integrated peak areas using genesis peak integration and 15-point smoothing with Xcalibur 2.0.7.
49
3.4. Results
3.4.1. BPA glucuronidation kinetics
BPA glucuronidation kinetics were best modeled using the Michaelis-Menten equation (Figure 3.3.). When incubated with pooled male and female HLMs, the Vmax for
BPA glucuronidation was determined to be 4.71 ± 0.30 nmol/min/mg protein, and the Km was 45.8 ± 8.9 μM.
3.4.2. Genistein glucuronidation kinetics
The enzyme kinetics of genistein glucuronidation were best fit using the
Michaelis-Menten model. When data from all three replicates were pooled together, the mean Vmax for genistein glucuronidation by HLMs was 0.93 ± 0.10 nmol/min/mg protein.
When co-incubated with 25 μM BPA, the mean Vmax for genistein glucuronidation significantly decreased to 0.62 ± 0.05 nmol/min/mg protein. Km values remained relatively unchanged between samples incubated with HLMs in the absence (15.1 ± 7.9
μM) and presence (21.5 ± 7.7 μM) of 25 μM BPA. (Figure 3.4.A) When genistein glucuronidation was performed with pooled male and female RLMs, mean Vmax values of
2.91 ± 0.26 and 3.05 ± 0.41 nmol/min/mg protein were achieved in the absence and presence of 25 μM BPA, respectively, reported from the pooled data of all three replicates. Km values for genistein glucuronidation by RLMs were 49.4 ± 14 μM in the absence of BPA and 84.0 ± 28 μM in the presence of 25 μM BPA (Figure 3.5.A). 50
Lineweaver-Burk plots were constructed to determine Ki values as well as the type of enzyme inhibition, on genistein glucuronidation by BPA. According to the
Lineweaver-Burk plots from the pooled replicate data, BPA exerted non-competitive inhibition for genistein glucuronidation in HLMs with a Ki value of 58.7 μM, and competitive inhibition for genistein glucuronidation in RLMs, with a Ki value of 35.7 μM
(Figure 3.4.B and Figure 3.5.B, respectively). Variation in Vmax, Km, and Ki values among replicates was minimal for both HLMs and RLMs (Figures 3.6., 3.7., 3.8., 3.9.,
3.10., and 3.11.).
The effects of varying concentrations BPA on glucuronidation activity in HLMs are shown in Figure 3.12. The IC50 value of BPA for genistein glucuronidation in HLMs was 37.0 μM.
51
3.5. Discussion
The work presented here shows that BPA inhibits the glucuronidation activity of genistein in both human and rat liver microsomes. The glucuronide is the predominant metabolite of genistein formed in both humans and rats. Inhibition of genistein’s glucuronidation therefore increases the bioavailability of genistein in the presence of
BPA, thus allowing genistein to have a prolonged pharmacological effect. Extended biological action of genistein could exacerbate the compound’s adverse effects, especially among populations that are particularly vulnerable to the endocrine disrupting effects of the natural phytoestrogen, such as neonates. Such an alteration in metabolic capacity is noteworthy, considering that both humans and laboratory rodents are routinely exposed to BPA and genistein simultaneously in their everyday lives (Thigpen et al,
2004; Patisaul and Jefferson, 2010).
It is interesting that the type of UGT enzyme inhibition elicited by BPA is different in HLMs and RLMs. In HLMs, the presence of BPA induced a significant decrease in Vmax, while Km remained unaffected. These characteristics, along with the
Lineweaver Burk plot for co-exposures to genistein and BPA in HLMs are suggestive of non-competitive enzyme inhibition (Figure 3.4.B). On the other hand, increased Km values and unchanged Vmax values in the presence of inhibitor are hallmarks of competitive inhibition. These trends, coupled with the Lineweaver Burk plots obtained from incubations of genistein and BPA with RLMs suggest competitive inhibition
(Figure 3.5.B).
The disparity in UGT inhibition type caused by BPA between species may be due to a variety of reasons. One possible explanation for the difference in inhibition type of 52 genistein glucuronidation observed between human and rat liver microsomes is that different UGT isoforms may be responsible for genistein’s metabolism in the different species. It is reasonable to consider that UGT isoforms are present at different levels in
HLMs than in RLMs, thus contributing to different glucuronidation capacities between the two species.
It is also possible that BPA may selectively inhibit a particular UGT isoform that is responsible for the glucuronidation of genistein. Evidence of BPA selectively inhibiting enzyme activity has been reported; previous work shows that BPA inhibits the phase I metabolism of a variety of xenobiotics via specific inhibition of particular cytochrome p450 enzymes in rat liver microsomes (Hanioka et al., 2000). Additionally, it has also been reported that BPA may inhibit the activity of human UGT 1A6, which is one of the key isoforms responsible for the metabolism of genistein in humans (Hanioka et al, 2008b), although this has not been tested in our laboratory. This interaction could be implicated in the altered metabolism of genistein observed during binary exposures in
HLMs.
In vitro analyses offer distinct advantages, but they are also limited in their utility.
Xenobiotic incubations with liver microsomes provide a valuable model for rapid screening of drug-to-drug interactions. Since the majority of predominant UGTs involved in the metabolism of BPA (rat UGT 2B1, human UGT 2B15) and genistein
(UGTs 1A1, 1A6, and 1A9) are expressed in the liver, the glucuronidation of genistein in the absence and presence of BPA can be easily assessed using liver microsomes. Overall metabolic pathways, however, cannot be investigated using liver microsomes, due to their inherent limited capacity for sulfation. Hepatocytes should be used instead of liver 53 microsomes to assess alterations of more global metabolism. Finally, studies using liver microsomes obviously exclude the analysis of enzymes that are expressed extra- hepatically, whereas in vivo studies allow for the analysis of the contribution of extra- hepatic as well as hepatic enzymes in the metabolism of xenobiotics.
Potential human risk from exposures to xenobiotics is often assessed by data generated from animal models. Such a method is often sufficient, since the animal data frequently mirrors the human data. In some circumstances, however, data generated from laboratory animals does not directly parallel the effects observed in humans, and alternate models are then required to predict risk to human health. The data presented here offer such an example of an incidence where animal data and human data do not match at the levels of genistein and BPA tested, and therefore human risk assessment for co-exposures to genistein and BPA should not necessarily be based upon data generated from rat models. 54
3.6. Conclusion
BPA induced non-competitive inhibition for genistein glucuronidation in HLMs, with a Ki value of 58.7 μM, and competitive enzyme inhibition for genistein glucuronidation in RLMs, at a Ki of 35.7 μM. Altered metabolism of genistein in the presence of BPA may impact the phytoestrogen’s toxicity.
Portions of Section 3.0. have been submitted for publication in Drug Metabolism and
Disposition, ID: DMD/2011/042366
55
4.0. GENERAL DISCUSSION
The work presented has established a validated method for the analysis of two common endocrine disrupting compounds genistein and BPA as well as their primary metabolites, in addition to having shed light on important metabolic interactions between the two compounds in human and rat liver microsomes during co-exposures.
The development of an analytical method to simultaneously extract and detect genistein, genistein glucuronide, BPA, and BPA glucuronide provides a means for improved efficiency in the determination of the four analytes of interest. The comprehensive method presented here allows for a single, comprehensive analysis instead of multiple analyses performed for each of the different analytes. This all- inclusive method requires fewer samples to be processed, thus saving time involved in sample preparation, clean-up via solid phase extraction, LC/MS injections, and data analysis. With fewer samples needing to be processed, fewer supplies and reagents are required, and less time dedicated by laboratory personnel is needed, all of which save money. Fewer analyses also means that smaller sample volumes may be collected.
Reduced requirements for sample size are especially beneficial when performing time- course studies, which involve frequent collections of biological samples, often in minute quantities.
Analyses of the results from the UGT inhibition studies using co-exposures to endocrine disrupting compounds genistein and BPA indicate that BPA inhibits the glucuronidation of genistein via non-competitive inhibition in human liver microsomes, and via competitive inhibition in rat liver microsomes. These findings are important for a 56 number of reasons. Because genistein’s glucuronidation is inhibited in the presence of
BPA, the bioavailability of genistein may be increased if sulfation and excretion rates remain constant, thus allowing for prolonged pharmacological action. Due to the critical window of exposure with genistein, prolonged action could result in enhanced endocrine disruption, especially in susceptible individuals such as developing fetuses and neonates.
Moreover, the data suggest that human livers metabolize genistein at different rates in the presence and absence of the environmental endocrine disruptor BPA, further contributing to possible altered toxicity.
Data generated from the inhibition studies also suggest that BPA inhibited glucuronidation of genistein by different types of inhibition in human and rat liver microsomes. Such differences have implications in risk assessment. In the absence of human studies, many predictions of human health risks posed by xenobiotics are assessed via extrapolation of data generated from experimentation with animal models, particularly rodent models. In the present case, one would likely not predict BPA to non- competitively inhibit the glucuronidation of genistein in human liver microsomes, based on the lack of such inhibition observed in rat liver microsomes. Making such an assumption would be hasty, as well as incorrect in this situation.
Additionally, previous studies have found that more catechols are formed in the metabolism of genistein by human hepatocytes than by rat hepatocytes (Bursztyka et al.,
2008a). Since catechols can form quinones and subsequently lead to the generation of
DNA adducts and carcinogenesis, genotoxicity and carcinogenicity analyses performed with rats, either in vivo or in vitro, may underestimate the true potential risk to humans.
It is obviously difficult to perform extensive in vivo studies in humans due to ethical 57 reasons, but when in vitro human data is possible to generate, it can prove to be immensely beneficial in providing directly relevant information upon which one could rationally base the assessment of human health risks.
The work presented here is not only important for its implications to risk assessment, but also because it addresses mixtures of compounds, which is an often overlooked aspect of toxicology. Despite their utmost relevance to human health, simple and complex chemical mixtures are grossly underrepresented within current toxicological literature, with nearly 95% of all resources, energy, and talents of scientific organizations being spent on the analysis of pure, single compounds (Yang, 1994). Realistically, most environmental and occupational human exposures do not involve single, pure chemicals in isolation, but rather include a large variety of different compounds in various media via numerous routes of exposure. Since most organisms live the majority of their lives exposed to multiple chemicals simultaneously or sequentially, it is imperative to study chemical mixtures.
Mixtures of chemicals can have substantially different biological effects than each individual chemical would in isolation. Because many individual xenoestrogens have very low estrogenic potencies compared to 17β-estradiol, it is often difficult to accurately predict potential human health risks for single compounds; however, it is very possible that xenoestrogens may interact with one another in an additive or synergistic manner when present simultaneously or sequentially, thus enhancing their estrogenic potentials
(Kortenkamp and Altenburger, 1998). For example, genistein exerts additive effects with estradiol in vitro at both ERα and ERβ (Casanova et al., 1999). Additionally, co- exposures may alter the metabolism of each individual xenobiotic present, further 58 complicating their interactive effects, as observed with genistein and BPA. Data from the inhibition studies with genistein and BPA that are presented here propose that the clearance and possibly the toxicity of certain xenobiotics may be altered in the presence of other chemicals at the same time. There is an infinite number of combinations of different xenobiotics that could potentially exhibit metabolic interactions when present simultaneously, ultimately affecting their toxicity. The combination of genistein and
BPA was specifically examined for the simultaneous analyses presented here due to shared estrogenic activity, similar biological effects, common every-day co-exposures, and comparable routes of metabolism. According to the results of this work, genistein and BPA in fact exhibit metabolic interactions in co-exposure scenarios, justifying the investigation of such chemical mixtures. In order to bring a sense of practicality and relevance to toxicological research, it is necessary to analyze exposures to chemical mixtures, such as the simultaneous co-exposure to bisphenol A and genistein examined here.
59
4.1. Areas of further research
The difference in UGT inhibition type between HLMs and RLMs in the presence of BPA observed herein may be due to a number of reasons. To elucidate the mechanism(s) behind the species difference, additional studies are required.
The disparity observed between species could be due to a difference on the molecular level, perhaps a differential presence and/or level of expression of specific
UGTs between the human and rat liver microsomes. It is known that the presence and level of expression of different UGTs varies between members of different species. Rats and humans have similar, but not identical, expression of hepatic UGTs (Mackenzie et al., 2005; Tukey and Strassburg, 2000). It is possible that rats have a unique UGT capable of glucuronidating genistein, that can be competitively inhibited by BPA, whereas multiple UGTs may be responsible for genistein’s glucuronidation in humans, each of which may be susceptible to enzyme inhibition, including but not limited to non- competitive inhibition by BPA. The presence and specific level of basal expression as well as activity of different UGTs in human and rat liver microsomes could be examined to explore species differences.
It has been previously reported that BPA inhibits the activity of human UGT1A6 in vitro via mixed competitive and non-competitive enzyme inhibition with experimentation using substrates serotonin and 4-methylumbelliferone (Hanioka et al.,
2008b). Genistein is known to be a substrate for human UGT1A6 (Doerge et al., 1999;
Lui et al., 2007). To investigate the specific role of UGT1A6 in the metabolism of genistein, and its altered activity in response to BPA, inhibition studies similar to those 60 presented here should be performed, substituting liver microsomes with isolated
UGT1A6 protein.
Finally, it is possible that the non-competitive inhibition observed in human liver microsomes may not be replicated in in vivo human studies. Rates of substrate glucuronidation observed in microsomes do not always accurately predict whole cell or in vivo glucuronidation capacities (Liu et al., 2007). Intact organisms are often endogenously equipped with compensatory mechanisms that are activated when a given pathway is inhibited or saturated. These compensatory mechanisms frequently require the use of feedback loops that are present in vivo, but are often disrupted or incomplete in vitro. Studies using intact cellular systems such as hepatocytes and organ cultures of whole livers may serve as useful models to investigate the inhibitory effects of BPA on genistein’s glucuronidation. In vivo experimentation would be especially useful to more accurately determination glucuronidation rates and to identify any such potential compensatory mechanisms that could thereby prevent the inhibition of genistein’s glucuronidation observed in human liver microsomes when BPA is present.
61
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Table 1.1. Estrogenic potentials, as measured by relative binding affinity (RBA) and dissociation constant (Ki) of 17 β-estradiol, genistein, and BPA to estrogen receptor (ER)-α and ER-β. Data summarized from Kuiper et al., 1997.
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Table 2.1. Summary of mass spectrometric parameters. Data was acquired in SRM mode with an isolation width of 2 m/z for each analyte.
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Table 2.2. Retention times for analytes of interest shift based on the HPLC column that is employed. Elution times (in minutes) for BPA, BPA gluc, genistein, and genistein gluc are reported for both HPLC columns tested, Kinetex C18 and Discovery C8. 73
Figure 1.1. Chemical structures of genistein (A) and genistein glucuronide (B).
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Figure 1.2. Chemical structure of 17β-estradiol.
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Figure 1.3. Chemical structures of BPA (A) and BPA glucuronide (B).
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Figure 1.4. Genistein and BPA co-exposure scenario. BPA-laden polycarbonate plastic baby bottle filled with soymilk, a rich source of genistein, provides a classic exposure scenario to young children—a population that is particularly vulnerable to the effects of endocrine disrupting compounds.
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Figure 2.1. Chromatograms of BPA (A) and genistein (B) from Kinetex C18. Elution times are 6.99 minutes for BPA and 3.75 minutes for genistein. Mass spectra of BPA (C) and genistein (D) were acquired in SRM mode with isolation width of 2 m/z. 78
Figure 2.2. Chromatograms of BPA (A), BPA gluc (A), genistein (B), and genistein gluc (B) from Discovery C8. Elution times of BPA, BPA gluc, genistein, and genistein gluc are 4.0, 1.6, 2.5, and 1.7 minutes, respectively. Mass spectra of BPA (C) and genistein (D) were acquired in SRM mode with isolation width of 2 m/z.
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A 140 120 A 100
80
60
40
Percent Recovery of BP of Recovery Percent 20
0 EM EA MA EMA 140 B 120 n 100
80
60 Bond Elut Plexa 40 Oasis HLB Percent Recovery of Genistei 20 UCT C18 0 EM EA MA EMA
Figure 2.3. Average recovery of BPA (A) and genistein (B) via SPE performed with different elution paradigms (EM: ethyl acetate, methanol; EA: ethyl acetate, acetonitrile; MA: methanol, acetonitrile; EMA: ethyl acetate, methanol, acetonitrile) on three different cartridge types (Bond Elut Plexa, Oasis HLB, and UCT C18). Optimal recovery of BPA and genistein is achieved with Bond Elut Plexa cartridges and EMA (98.1% ± 1.8 and 94.9% ± 8.0, respectively).
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Figure 2.4. Optimized SPE conditions (Bond Elut Plexa cartridge and EMA) yield average recoveries for BPA, BPA gluc, genistein, and genistein gluc of 98.1% ± 1.8, 91.4% ± 6.1, 94.9 ± 8.0, and 103 ± 6.1, respectively.
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Figure 3.1. Percent glucuronidation of genistein by human liver microsomes using 0.100, 0.050, 0.025, and 0.0125 mg protein/incubation during 30 minute incubations at 37°C. Ideal protein concentration for optimized conditions should result in 10-15% glucuronidation in order to minimize dramatic reduction of substrate concentration, which could cause increased variability of Vmax and Km values for each incubation. Based on these results, an HLM protein concentration of 0.0125 mg/incubation was identified to yield optimized conditions.
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Figure 3.2. Percent glucuronidation of genistein by rat liver microsomes using 0.0125, 0.09375, 0.00625, and 0.003125 mg protein/incubation during 30 minute incubations at 37°C. Ideal protein concentration for optimized conditions should result in 10-15% glucuronidation in order to minimize dramatic reduction of substrate concentration, which could cause increased variability of Vmax and Km values for each incubation. Based on these results, an RLM protein concentration of 0.005 mg/incubation was identified to yield optimized conditions.
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Figure 3.3. Enzyme kinetic plots of BPA glucuronidation using pooled human liver microsomes. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). Vmax = 4.71 ± 0.30 nmoles/min/mg and Km = 45.8 ± 8.9 μM.
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Figure 3.4. Enzyme kinetic plots of genistein glucuronidation (pooled data) using pooled human liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. (A) Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 0.93 ± 0.10 nmoles/min/mg and Km = 15.1 ± 7.9 μM. In presence of BPA, Vmax = 0.62 ± 0.05 nmoles/min/mg and Km = 21.5 ± 7.7 μM.
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Figure 3.5. Enzyme kinetic plots of genistein glucuronidation (pooled data) using pooled rat liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 2.91 ± 0.26 nmoles/min/mg and Km = 49.4 ± 14 μM. In presence of BPA, Vmax = 3.05 ± 0.41 nmoles/min/mg and Km = 84.0 ± 28 μM.
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Figure 3.6. Enzyme kinetic plots of genistein glucuronidation (rep 1) using pooled human liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 1.13 ± 0.05 nmoles/min/mg and Km = 11.4 ± 2.6 μM. In presence of BPA, Vmax = 0.69 ± 0.03 nmoles/min/mg and Km = 13.8 ± 2.4 μM.
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Figure 3.7. Enzyme kinetic plots of genistein glucuronidation (replicate 2) using pooled human liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 0.89 ± 0.06 nmoles/min/mg and Km = 24.0 ± 6.0 μM. In presence of BPA, Vmax = 0.57 ± 0.04 nmoles/min/mg and Km = 30.8 ± 7.1 μM.
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Figure 3.8. Enzyme kinetic plots of genistein glucuronidation (replicate 3) using pooled human liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 0.91 ± 0.04 nmoles/min/mg and Km = 21.1 ± 3.6 μM. In presence of BPA, Vmax = 0.60 ± 0.03 nmoles/min/mg and Km = 24.9 ± 5.5 μM.
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Figure 3.9. Enzyme kinetic plots of genistein glucuronidation (replicate 1) using pooled rat liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 2.83 ± 0.33 nmoles/min/mg and Km = 63.0 ± 20 μM. In presence of BPA, Vmax = 2.68 ± 0.14 nmoles/min/mg and Km = 82.9 ± 11 μM.
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Figure 3.10. Enzyme kinetic plots of genistein glucuronidation (replicate 2) using pooled rat liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 3.14 ± 0.26 nmoles/min/mg and Km = 58.3 ± 14 μM. In presence of BPA, Vmax = 3.57 ± 0.34 nmoles/min/mg and Km = 96.5 ± 22 μM.
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Figure 3.11. Enzyme kinetic plots of genistein glucuronidation (replicate 3) using pooled rat liver microsomes in the absence (0 μM; open circles) and presence (25 μM; closed circles) of BPA. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B). In absence of BPA, Vmax = 2.79 ± 0.13 nmoles/min/mg and Km = 33.28 ± 5.4 μM. In presence of BPA, Vmax = 3.59 ± 0.37 nmoles/min/mg and Km = 74.6 ± 20 μM.
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Figure 3.12. The effects of BPA at concentrations ranging from 0-250 µM on the glucuronidation of genistein in pooled male and female HLMs. The IC50 value of BPA for genistein glucuronidation in HLMs is 37.0 μM.