Epigenetics,+Neurobehavior,+and+Toxic+Stress+in+Children+with+Prenatal+ Methamphetamine+Exposure:+An+Introduction+ + &+ Epigenetics+of+Neurobehavior+in+Children+with+Prenatal+Methamphetamine+ Exposure+ ! Written+By+ Oluwadamilola+Oluwadolapo+OniGOrisan+ B.A.,+Yale+University,+2013+ + + Thesis&submitted&in&partial&fulfillment&of&the&requirements&for&the&& Degree&of&Master&of&Public&Health&at&the Brown&University&School&of&Public&Health& Providence,&Rhode&Island May+1,+2017 ! ii! This thesis by Oluwadamilola Oni-Orisan is accepted in its present form by the Brown University School of Public Health as satisfying the thesis requirements for the degree of Master of Public Health Date:________________ ___________________________ Barry Lester, Ph.D., Advisor Date:________________ ___________________________ Deborah Pearlman, Ph.D., Reader Date:________________ ___________________________ Patrick M. Vivier, M.D.,Ph.D. Director, Master of Public Health Program Approved by the Graduate Council Date:______________ _____________________________ Andrew Campbell, Dean of the Graduate School ! iii! Vita Oluwadamilola “Dami” Oni-Orisan was born on January 23rd, 1992, to Tutu Oni-Orisan and Bolaji Oni-Orisan. Three short years later, her younger brother Tobi Oni-Orisan was born. Dami attended both the Greater Hartford Academy of Mathematics & Science (a specialized half-day magnet school for advanced coursework) and New Britain High School simultaneously, splitting her extended school day between the two schools and ultimately graduating as the Valedictorian of both in 2009. She then attended Yale University, where she pursued a double major and graduated with her Bachelors in Molecular, Cellular & Developmental Biology and Portuguese in 2013. At Yale, she immersed herself in activities concerning health and education disparities by leading international development service trips, teaching in New Haven public schools, and volunteering at the HAVEN Free Clinic. As an undergraduate student she immersed herself in Lusophone studies and spent significant time abroad in Brazil and Cape Verde, visiting both countries twice before graduating. After graduating, she continued to pursue her professional interests in eliminating disparities. Dami worked as the Residential Director for Yale’s Science Collaborative Hands-On Learning and Research (SCHOLAR) Program for high school students. She was then awarded an international healthcare-education fellowship to travel to the Açores (Portugal), where she interned at the Ministry of Health and taught English at the Base 5 English Academy. Soon after, she was selected to participate in an intensive infectious disease epidemiology course at the prestigious Faculdade de Medicina at the Universidade de São Paulo in São Paulo, Brazil. A few weeks after the course ended, Dami entered the Brown University School of Public Health to pursue her Master of Public Health degree with a focus in epidemiology and global health. Ever passionate about working in Brazil, Dami was awarded the NIH Minority Health International Research Training Fellowship to conduct infectious disease research at the Universidade Federal da Bahia in Salvador, Brazil during the summer after her first year of study (2016). She was also one of a select group of graduate students awarded the University’s Green Grant for Research in Brazil (2016). Upon her return to Brown that fall, Dami was named an inaugural Rhode Island Leadership Education in Neurodevelopmental Disabilities (RI-LEND) Fellow, for which she completes interdisciplinary training at the Children’s Neurodevelopment Center at Hasbro Children’s Hospital. Dami has thoroughly enjoyed conducting research at the Center for the Study of Children At-risk (Women & Infants’ Hospital) in Providence. Dami looks forward to continuing to pursue her passions for global health and child neurodevelopment after graduation. ! iv! Preface and acknowledgments This thesis would not have been possible without the tremendous support of Dr. Barry Lester, Dr. Deborah Pearlman, and Lynne Dansereau. Dr. Lester, thank you for accepting my thesis proposal and allowing me to join your research team. I truly appreciate your mentorship, research training, and support of my professional endeavors. Dr. Pearlman, thank you for your guidance and critical feedback during the process of completing my thesis. You have provided insightful advice and a listening ear ever since I enrolled at Brown. Lynne, thank you for your incredible support in teaching me about the IDEAL dataset and reviewing my work. Your kindness helped make this an enjoyable experience. Additionally, I would like to thank Lee Breault for her inspirational conversations and help scheduling my meetings with Dr. Lester and Lynne. Thank you for always accommodating me and welcoming me. I would also like to thank Joann Barao. Your personal emails and open door policy were warm gestures that show how much you care. Furthermore, I would like to thank Dr. Patrick Vivier. Your graciousness and advice were essential to the completion of my thesis. And to Dr. Simin Liu, thank you for your persistent support of me and my goals. You have been a great advisor and I value your counsel. Furthermore, I would like to thank my family for their love and support. Your confidence in me has brought me to this point. Last, but not least, I would like to thank God, the source of my strength. Through Him, all things are possible. ! v! TABLE OF CONTENTS PART I. LITERATURE REVIEW—EPIGENETICS, NEUROBEHAVIOR, AND TOXIC STRESS IN CHILDREN WITH PRENATAL METHAMPHETAMINE EXPOSURE: AN INTRODUCTION Introduction……………………………………………………………………………………… 1 Prenatal methamphetamine exposure……………………………………………………………..2 What is epigenetics………………………………………………………………………………..4 A history of epigenetics…….………………………………………………………..........6 Figure 1: DNA methylation during transcription, from Lester et al., 2016……….6 Epigenetics and the study of human behavior…………..………………….……………..7 Epigenetics and embryonic development…………………………………………………9 Basic approaches to the study of epigenetics……………………………………………...9 Why look at the epigenetics of prenatal methamphetamine exposure?.............................10 Figure 2: Epigenetic stress model diagram, from Lester & Padbury, 2009…...…11 Gene of interest…………………………………………………………………………………..12 Cortisol and toxic stress: life in the balance……………………………………………………..13 Conclusion…………………………………………………………………………………….....17 References………………………………………………………………………………………..18 PART II. DATA PAPER—EPIGENETICS OF NEUROBEHAVIOR IN CHILDREN WITH PRENATAL METHAMPHETAMINE EXPOSURE Abstract…………………………………………………………………………………………..26 Introduction……………………………………………………………………………………....27 Methods…………………………………………………………………………………………..29 Results……………………………………………………………………………………………32 Discussion……………………………………………………………………………………..…33 Conclusion……………………………………………………………………………….37 Table 1: The early adversity index accounted for cumulative measures of adversity from birth through age 5 years by calculating the sum of a set of binary indicators………………………..39 Table 2: Comparison of dyads included and not included……………………………………….40 Table 3: Maternal and neonatal characteristics by MA exposure………………………………..41 Table 4: Summary of multivariate analysis for predicting methylation at CpG2 of 11B- HSD2……………………………………………………………………………………………..42 References………………………………………………………………………………………..43 Oluwadamilola Oni-Orisan 1 PART I. Epigenetics, Neurobehavior, and Toxic Stress in Children with Prenatal Methamphetamine Exposure: An Introduction Introduction The study of human behavior through the lens of epigenetics is novel given the relatively recent development of epigenetic behavioral research. The application of epigenetics to behavioral research is of great significance given the vast potential there is to use epigenetics to ascertain the molecular processes that act on gene expression and how these processes shape behavior and development in children. Of great interest is understanding how prenatal methamphetamine exposure is potentially associated with epigenetic modification of the fetus’s neuroendocrine system. Can methamphetamine exposure in utero act as a stressor on the developing fetus, thereby altering normal neurobehavior? The purpose of this article is to review what is known about prenatal methamphetamine exposure, provide a brief overview of epigenetics (including DNA methylation), and discuss the motives for applying epigenetic analysis to study prenatal methamphetamine exposure. Thereafter, we will discuss the selection of 11-Beta-hydroxysteroid dehydrogenase Type II as the gene of interest in this application and the use of cortisol as a biomarker of toxic stress. Table of Contents: I.! Prenatal methamphetamine exposure II.! What is epigenetics? a.! A history of epigenetics b.! Epigenetics and the study of human behavior c.! Epigenetics and embryonic development d.! Basic approaches to the study of epigenetics e.! Why look at the epigenetics of prenatal methamphetamine exposure? III.! Gene of interest IV.! Cortisol and toxic stress: life in the balance Oluwadamilola Oni-Orisan 2 I.! Prenatal Methamphetamine Exposure Methamphetamine use in the United States is a major public health concern that continues to grow (Substance Abuse and Mental Health Services Administration—SAMHSA, 2015). In 2010, the SAMHSA National Surveys on Drug Use and Health estimated that 353,000 people (0.1% of the national population) used methamphetamine in the month before the survey was administered (SAMHSA, 2011); in 2012, this figure rose to 440,000 people (0.2% of the national population) (SAMHSA, 2013). A year later, the upward trend in methamphetamine use continued; SAMHSA reports that in 2013, 595,000 people (0.2% of the national population) used methamphetamines in the month before the survey (SAMHSA, 2014). In 2014, 0.1% of pregnant women used methamphetamine (SAMHSA, 2015). In 2015, 4.7% of pregnant women aged 15- 44 years used illicit drugs including methamphetamine (SAMHSA, 2016). The search for prevalence of methamphetamine use among pregnant women exposed a glaring gap in the literature given that the most recent estimates are out of date. Between 2002 and 2004, pregnant women who used methamphetamines constituted a sizeable population in the United States; an estimated 38,000 women belonged to this group (Colliver et al., 2006). Of the pregnant women enrolled in federally funded substance abuse treatment programs in 2008, 24% were admitted for methamphetamine use (Terplan et al., 2009). Although current estimates for the prevalence of methamphetamine use among pregnant women are not available, a review of the aforementioned national trends suggests that it is reasonable to infer that drug abuse in this key population has also increased. Methamphetamine is classified as a stimulant and a neurotoxin that is known to act on both the central nervous system (which consists of the brain and spinal cord) and the sympathetic nervous system (which controls arousal) (Davidson et al., 2001). While methamphetamine use is associated with a host of adverse health outcomes in the general population of users (Berman et Oluwadamilola Oni-Orisan 3 al., 2008; Block et al., 2002; Chang et al., 2007; Sommers et al., 2006), pregnant women who use methamphetamines are a particularly important subset of the population to study given the risk of associated developmental outcomes in their children (Abar et al., 2012). Infants born with prenatal methamphetamine exposure often suffer from neurobehavioral disinhibition (ND) (Tarter et al., 2003; Abar et al., 2012), including cognitive and behavioral deficits (Lester & Lagsse, 2010; Liles et al., 2012; Paz MS et al, 2008; Eze et al., 2016; LaGasse et al., 2012; Wouldes et al., 2004), altered brain architecture, and imbalanced neurochemistry (Sowell et al., 2010). These detrimental effects have been shown to extend into memory (Chang et al., 2004; Lu et al., 2009; Piper et al., 2011), attention (Chang et al., 2004), inhibitory control (Derauf et al., 2012), motor control (Chang et al., 2009; Smith et al., 2011), and vision (Chang et al., 2009). The results of a 2010 study found that children with prenatal methamphetamine and alcohol exposure had significantly smaller brains than children who had been exposed to alcohol only, methamphetamine only, or neither toxin, indicating that prenatal methamphetamine exposure could be unique from other teratogens in terms of the danger that it poses to the developing fetus (Sowell et al., 2010). Studies have shown that these outcomes are associated with an increased risk of substance use/abuse later in life (Chapman et al., 2007; Lester et al., 2012; McNamee et al., 2008; Tarter et al., 2003). Oluwadamilola Oni-Orisan 4 II.! What is epigenetics? Epigenetics is the study of environmental and experiential factors that influence gene expression and, consequentially, development (Moore, 2015). The human body is composed of cells, each of which is characterized by its constituent proteins (Bird, 2002). DNA serves as the body’s blueprint for building protein by providing the basic code that determines protein structure through two important mechanisms: transcription and translation. A strand of DNA spells out a genetic code that is composed of four possible chemical bases. Much like the English alphabet is composed of 26 letters, the genetic alphabet (or code) is composed of four bases. These four bases are adenine (A), guanine (G), cytosine (C), and thymine (T). Similar to the fact that letters in the English alphabet can be combined to spell words and construct sentences, the four bases can be organized into sequences called strands of DNA. The double helix structure of DNA results when two complementary strands of DNA bind to each other. This binding occurs when complementary base pairs are formed between adenine and thymine, or cytosine and guanine, respectively. Unlike genetic mutations, which can inhibit binding between appropriate bases or even delete bases entirely, epigenetic processes do not alter the presence or binding of the four base pairs. Epigenetic processes have no effect on the structure of DNA (Russo et al., 1996). Epigenetic processes alter the way that DNA is expressed. Thus, in referring back to the English language analogy, while epigenetic processes do not alter the spelling of a word, the epigenetic influence is analogous to the difference in accent between an Irishman, an Englishman, an American, and an Indian—all four can read the same sentence, but their pronunciation will differ in terms of accented syllables and seemingly silent letters. Much like an accent influences the degree to which letter sounds may be emphasized or dropped altogether, epigenetics mediates the degree to which a gene is expressed (or whether it is expressed at all). Oluwadamilola Oni-Orisan 5 The various exposures that a living being encounters during the life course can shape gene expression through epigenetic processes that influence the expression of DNA without modifying the DNA structure (Griffiths & Hunter, 2014). These processes take place when a sequence of DNA is transcribed into ribonucleic acid (RNA), which is then translated into protein. The chemical bases of the DNA strand lay the foundation upon which the body builds protein. Thus, the first step in creating protein from the genetic code is to use the bases as a template to transcribe DNA into RNA; this process is appropriately called “transcription”. Once DNA is transcribed into RNA, RNA serves as the template for building protein. Thus, in the second step RNA is effectively translated into protein via the process of “translation”. DNA methylation is the epigenetic mechanism that occurs before transcription, and is the primary focus of epigenetic studies in humans. The second process, histone modification, occurs after translation and is yet to be successfully applied to the study of epigenetics in humans. The most commonly studied avenues of histone modification are methylation, acetylation, and phosphorylation (Jiang et al., 2008; Hunter, 2012). DNA methylation is the epigenetic mechanism that occurs during pre-transcription of DNA into RNA. This process results in the methylation, or addition of a methyl group, to a cytosine base on the DNA strand (Zhang et al., 2005). As a result, in spite of the DNA sequence of bases remaining unchanged, the problem then arises in transcription when the bulkiness of the methyl group attached to the DNA’s cytosine may prevent the sequence from being properly transcribed into RNA. This interruption in transcription is illustrated below in Figure 1 (Lester et al., 2016). Oluwadamilola Oni-Orisan 6 Figure 1. DNA methylation during transcription, from Lester et al., 2016. Methylation usually occurs on a cytosine base (Griffiths & Hunter, 2014) that is adjacent to a guanine on the same DNA strand; such a cytosine is referred to as a cytosine-phosphate- guanine (CpG) site (Lester et al., 2016). CpG sites are often concentrated at the promoter end of the DNA strand within CpG islands (Griffiths & Hunter, 2014), which is where the transcription process begins, but can also be found in exons and introns of genes (Gelfman et al., 2013). A history of epigenetics Greek philosopher Aristotle is credited with coining the term “epigenesis”, which would eventually serve as the precursor to the modern term “epigenetics” (Lester et al., 2016). Aristotle used the term to refer to the sequential nature of development in accordance with a series of phases. Centuries later, scientists Spemann and Mangold would revisit the topic in 1924 and propose the theory that cells can be activated and deactivated. The final step in the naming process came less than 20 years later when Conrad Waddington officially created the term Oluwadamilola Oni-Orisan 7 “epigenetics” to refer to the dynamic relationship between nature and nurture, asserting that genetic expression can be modified by environmental exposures. In his words, epigenetics is “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Waddington, 1942). Thus, a field was born. Nonetheless, although Waddington officially named the field of epigenetics in 1942 (Waddington, 1942), it is only recently that scientists have begun to use epigenetic principles to explain human behavior. Epigenetics and the study of human behavior Behavioral epigenetics is the science of understanding how a person’s life experience (including diet, exposure to toxins, and parenting style of the individual’s parents) influences how his/her genes function (Moore, 2015). Given the novelty of the study of human behavior in the context of epigenetics, there are many questions yet to be answered. Among them are: •! Is there an association between epigenetics and behavior? Is there a relationship between the two at all? •! How much methylation of a gene is required to produce changes in behavior? •! Is the methylation cut-off point the same for all genes, or is the amount of methylation required to influence behavior specific to the gene at hand? •! Are all genes equally impacted by DNA methylation, or are the effects of methylation as observed in behavior more pronounced on some genes compared to others? In recent years, studies have been conducted in order to answer the questions above. Researchers recently concluded that the relationship between DNA methylation and human behavior is a bidirectional one; just as methylation of genes can influence behavior, behavior is now known to reciprocate the favor by influencing methylation (Knopik et al., 2012). Furthermore, scientists have concluded that the relationship between methylation and behavior is not a one-size-fits-all Oluwadamilola Oni-Orisan 8 dosage; the same degree of methylation can elicit different degrees of behavioral change in different genes. In other words, two genes may require very different amounts of methylation in order to promote the same behavior. Of particular intrigue is a question that remains unanswered: Could varying amounts of methylation across several genes account for individual differences in behavior (Lester et al., 2016)? Studies have shown that epigenetic changes can alter normal child development and behavior (Conradt et al., 2016; Conradt et al., 2013; Knopik et al., 2012; Lester et al., 2013; Lester et al., 2012, 2015; Montirosso et al., 2016). For example, researchers have found that prenatal and postnatal programming regulate epigenetic modifications in children to adjust to environmental adversity (Lester et al., 2014). That study found that infants at the highest risk for depression experienced maternal depression both pre- and post-partum, periods in which adversity could further aggravate depression. Fetal exposure to maternal depression during pregnancy results in elevated circulating cortisol, elevated norepinephrine, and decreased serotonin. The placental genes that regulate uptake of serotonin (SLC6A4) and control the neuroendocrine environment (NET, 11B-HSD2, NR3C1) reprogram the HPA axis, thereby modifying postnatal baseline levels of performance of the HPA axis and related physiological systems. This reprogramming results in increased cortisol secretion, decreased serotonin secretion, and thus a lowered threshold of HPA reactivity to the postnatal environment in anticipation of further exposure to adversity in the postnatal environment. After birth, the increased physiological reactivity (cortisol secretion) is protective against early adversity in the short-term, but makes the child more susceptible to long term chronic stress and the effects of maternal postpartum depression. Oluwadamilola Oni-Orisan 9 Epigenetics and embryonic development Little is known about the relationship between prenatal methamphetamine exposure and epigenetic changes in the neurobehavior of the developing fetus. Epigenetic changes occur in the developing embryo via fetal programming, behavioral intervention, or germline transmission (Griffiths & Hunter, 2014; Bohacek & Mansuy, 2013). Epigenetics is essential to directing normal development and promoting the stability of the genome, particularly during sensitive periods (Fan et al., 2005). For example, epigenetic changes drive the process of cell differentiation (Fan et al., 2005). After sperm fertilizes an egg, cells in the resulting fertilized embryo enter several cycles of cell division. DNA methylation of the genes in these cells directs their fate in different regions of the embryo. Methylation helps to program the fate of the cell by directing which genes are expressed and repressed, thereby characterizing the genetic identity of each cell. This genetic identity yields the cell’s phenotype, which ultimately produces the wide variety of cells needed to compose the different tissues and organ systems of the body during normal development. Basic approaches to the study of epigenetics The three primary approaches to the study of epigenetics are the candidate/target gene approach, the study of a set of genes involved in a single pathway, and the genome-wide approach. These three approaches differ in the scale of focus. While the candidate/target gene approach focuses on the study of a single gene in order to yield information about individual regions on said gene, a second approach is to collectively study multiple genes that are part of a common pathway (Lester et al., 2016). For those interested in studying a greater number of genes, it is possible to use microarray technology to examine thousands of gene or even the full genome (Maccani et al., 2015). Thus, each level of study has its use, and no one method trumps Oluwadamilola Oni-Orisan 10 the others; instead, the combination of these methods is ideal in order to generate a full understanding of epigenetic interactions in a system (Lester et al., 2016). Why look at the epigenetics of prenatal methamphetamine exposure? An embryo’s exposure to prenatal stress is often profound because this stress can occur during a “critical period” in fetal development, a window of time in which stress causes irreversible epigenetic changes (Callaghan et al. 2013). Research has shown that DNA methylation and the critical period during which it occurs are associated with developmental outcome (Simmons et al., 2012, 2013); when prenatal stress takes place in a certain critical period, epigenetic changes can continue to shape the embryo’s development (Griffiths & Hunter, 2014). In studying the epigenetics of prenatal cocaine exposure, Lester and Padbury summarized the pathophysiology of cocaine along two known pathways (neurochemical and vasoconstrictive), and proposed a third pathophysiological pathway: the epigenetic stress model (Lester & Padbury, 2009), illustrated in Figure 2. Their hypothetical model postulated that cocaine, in addition to acting as a neurotransmitter blocker in the neurochemical pathway and promoter of plasma catecholamine concentrations in the vasoconstrictive pathway, could also act as a stressor that disrupts neuroendocrine homeostasis and normal fetal programming (Lester & Padbury, 2009). As depicted in Figure 2, such disruptions in the prenatal environment would result in decreased levels of key placental genes that program the fetal neuroendocrine environment in utero (Lester & Padbury, 2009). The validity of this model is yet to be tested in the context of prenatal cocaine use or any other prenatal drug exposure. Children with high PME have been found to have increased cortisol reactivity, a relationship modified by the experience of mild to moderate potential for child physical abuse (Kirlic et al., 2012). Increased cortisol reactivity has also been noted in children (Eiden et al., Oluwadamilola Oni-Orisan 11 2009) and adolescents (Chaplin et al., 2010) who were prenatally exposed to cocaine (Lester et al., 2010). Studies have also shown that early life stress (early adversity) can result in either increased or decreased cortisol reactivity, a relationship modified by chronicity and severity (De Bellis, 2005; Heim et al., 2000). Researchers have proposed that early adversity can induce irreversible epigenetic effects on certain genes that may increase a child’s susceptibility to neuroendocrine and behavioral dysfunction (Weaver et al., 2004; McGowan et al., 2009; Miller et al., 2007). Various studies also support the theory that early adversity controls HPA axis reactivity (Levine, 2005; Meaney et al., 2007; Hoffmann & Spengler, 2012; Heim et al 2008; Binder 2009; Morris 2012). The physiological effects of early adversity are important in studying PME because PME is associated with increased early adversity experienced by the child (Abar et al., 2012). An early IDEAL study examined the relationship between PME, early adversity, and neurobehavioral disinhibition and found that high levels of early adversity were associated with greater behavioral and emotional control problems at age 5 years (Abar et al., 2012). Figure 2. Epigenetic Stress Model Diagram illustrating the effects of in utero cocaine exposure on placental genes affecting neuroendocrine function and behavioral development from Lester & Padbury, 2009. Oluwadamilola Oni-Orisan 12 III.! Gene of Interest The two key placental genes depicted in Figure 2 are the norepinephrine transporter and 11-Beta-hydroxysteroid dehydrogenase Type II (11B-HSD2). In studying the potential role of prenatal methamphetamine exposure as a stressor, the gene 11B-HSD2 is of particular interest because it converts maternal cortisol (a biomarker of stress described in the next section) into its inert analog, cortisone, in order to protect the fetus from exposure to maternal cortisol (Lopez & Craft, 1981). As illustrated by gaps in the literature, the theoretical relationship between infant behavior and the epigenetic modification of the placental genes modeled in Figure 2 has not been studied. The rationale for studying 11B-HSD2, a glucocorticoid stress-related gene, is that it permits the study of methamphetamine as a stressor and how it modifies the expression of select candidate genes—in short, it allows researchers to test the hypothesis of the third pathophysiology (epigenetic stress model) and whether or not the pathway can be generalized beyond prenatal cocaine exposure. The gene 11B-HSD2 is a stress-responsive gene that codes for an enzymatic protein. This enzyme regulates the amount of active cortisol in the blood by a mechanism that deactivates cortisol; deactivated cortisol is referred to as cortisone (Jenkins & Sampson, 1966). During pregnancy, the enzyme protects the fetus from the mother’s cortisol by converting it into inactive cortisone (Murphy et al., 1974). Methylation of 11B-HSD2 at the promoter region has been shown to alter 11B-HSD2 gene expression (Jensen Peña et al., 2012). This faulty enzymatic function results in adverse effects such as prolonged HPA reactivity, low birth weight, greater anxiety, greater hyperglycemia, and greater hypertension in rodent models (Harris & Seckl, 2011). While in 2016, researchers concluded that methylation of 11B-HSD2 associated with depression yields adverse effects in humans (Conradt et al., 2016), studies have yet to show Oluwadamilola Oni-Orisan 13 whether or not 11B-HSD2 methylation is associated with prenatal methamphetamine exposure and what potential effects would arise from such an association. Furthermore, it is currently unknown whether or not increased methylation of the 11-Beta gene is associated with higher cortisone levels in children who were exposed to pre-natal methamphetamine as compared to those children who had no such exposure. IV.! Cortisol and Toxic Stress: Life in the Balance The hormone cortisol is one of the most commonly used stress biomarkers in pediatric research (Garner et al., 2011; Slopen et al., 2014). It is produced by the hypothalamic-pituitary- adrenal (HPA) axis. The HPA axis regulates the body’s neuroendocrine stress responses (Slopen et al., 2014) , which is initiated upon an individual’s perception of an immediate threat in his surroundings. This triggers the brain to release corticotropin-releasing factor (CRF) which prompts the pituitary gland to release adrenocorticotropic hormone (ACTH) (Gunnar & Vasquez, 2006) during what is commonly referred to as the “fight or flight response”. The cascade continues when ACTH in turn activates cells in the brain’s adrenal cortex to release cortisol into the bloodstream, thereby providing the adrenaline rush that propels one into action in a situation of duress. Under the conditions of a normal stress reaction, a process of negative feedback reduces the cortisol in the bloodstream. This process involves binding active glucocorticoids, such as cortisol, to glucocorticoid receptors in the brain. Once bound, cortisol is deactivated and transformed into its inactive form cortisone, thereby disinhibiting the production and release of CRF (and therefore cortisol) and ultimately deactivating the HPA axis (Bale & Vale, 2004; Koob et al., 1994; Plotsky et al., 1989). However, the normal response of the HPA axis to stress is interrupted when DNA methylation of NR3C1, the gene that codes for glucocorticoid receptors, occurs. This Oluwadamilola Oni-Orisan 14 methylation is disruptive because it inhibits the ability of the receptor to properly bind glucocorticoids such as cortisol in humans (Oberlander et al., 2008). As a result, the negative feedback mechanism that normally regulates active cortisol is modified; instead of binding active cortisol and stopping further production of it, active cortisol remains in the bloodstream. Thus, the brain never receives the signal that it needs to reduce cortisol production. The HPA axis remains activated. This prolonged activation of the stress response system can result in toxic stress, the most dangerous stress response (Shonkoff et al., 2011). Toxic stress is dangerous because it is believed to disrupt brain circuitry normal organ and metabolic systems during sensitive development periods, which can produce physiological changes that lead to learning and behavior impairments, and physical and mental illness (Shonkoff et al., 2011). Research has shown that exposure to adversity in early childhood can compound as toxic stress, serving as a hazard to a child’s healthy growth and development during the life course (O’Malley et al., 2015). Adversity in early childhood, also referred to as early life stress, is a key exposure of interest in children’s behavioral research because it accounts for certain factors in a child’s environment. Studies have shown that early life stress can also result in either increased or decreased cortisol reactivity, a relationship modified by chronicity and severity (De Bellis et al., 2005; Heim et al., 2000). In evaluating the potential association between environment and neurobehavior, scientists should focus on epigenetics rather than genotype. Epigenetic changes are modifiable by early life stress (the environment) while genotype is not. The HPA axis acts on three major systems: the central nervous system, the metabolic system, and the immune system. The central nervous system is responsible for regulating an individual’s emotional state, learning comprehension, and memory (Slopen et al., 2014). The metabolic system manages glucose storage and directs glucose usage. Lastly, the immune system Oluwadamilola Oni-Orisan 15 regulates the body’s inflammatory response and the development of lymphocytes, white blood cells that protect the body against foreign invaders. Of particular interest to this research is the impact of toxic stress on neurobehavior, which is regulated by the central nervous system. Human cortisol levels are known to follow the circadian rhythm, meaning that they are influenced by the time of day and normally peak in the morning between 7:00 am and 8:00 am (Kalsbeek et al., 1996). Scientists have proposed that this peak in cortisol serves to prepare the body for stresses associated with waking up, such as a demand for energy (Dickmeis et al., 2009; Kalsbeek et al., 1996). Cortisol levels increase steadily during sleep in anticipation of engagement in increased activity upon waking (Dickmeis et al., 2009; Kalsbeek et al., 1996). Cortisol can be measured in a variety of ways. Measurements of hair cortisol are collected in order to assess cumulative cortisol, as the literature asserts that hair contains the best cumulative store of cortisol for stress analysis (Garner et al., 2011). This is in stark comparison to salivary cortisol tests which account for short-term temporal variations more reflective of the immediate stress profile. Salivary cortisol concentration is a reliable indicator of the free cortisol concentration in plasma (Laudat et al., 1988). Both salivary and plasma cortisol levels can be assessed using Cortisol ELISA, a commercial enzyme-linked immunosorbent assay (Cortisol ELISA, IBL International, Hamburg, Germany). Kirlic et al. assessed cortisol stress reactivity with saliva samples by modifying a well- established method that induces stress in children (Gunnar et al., 2009; Jacobson et al., 1999). In this process, a baseline saliva sample was collected and the child’s emotional state rated upon completion of the Early Social Communication Scales (ESCS; Mundy et al., 1996). The ESCS is a 20-minute interaction task between the examiner and the child that codes for joint attention, social interactions, and behavioral regulation. The ESCS was followed by 12 minutes of both Oluwadamilola Oni-Orisan 16 structured and unstructured play between the child and caregiver, after which the child was left alone for 2 minutes at most. Then, the examiner returned to the child for 1 minute of no interaction before the caregiver returned. The caregiver returned earlier if the child became upset. The second saliva sample, which measured the stress response, was collected 20 minutes after the separation. Two polyurethane foam collectors were used to collect 0.5 mL samples for 1 minute. The samples were then stored at -20° C and stored until analysis. A high-sensitivity salivary cortisol enzyme immunoassay was used to analyze the samples (Kirlic et al., 2012). Increased cortisol reactivity has been noted in children (Eiden et al., 2009) and adolescents (Chaplin et al., 2010) who were prenatally exposed to cocaine. Children with high prenatal methamphetamine exposure have been found to have increased cortisol reactivity, a relationship modified by the experience of mild to moderate potential for child physical abuse (Kirlic et al., 2012). In lieu of cortisol, cortisone is another useful biomarker for measuring current stress (Vanaelst et al., 2013). Salivary cortisone has been reported to accurately reflect free serum cortisol during the stimulation of the adrenal glands (Perogamvros et al., 2010). In a study comparing the response patterns of cortisol and cortisone, researchers found that the two biomarkers exhibited a similar circadian rhythm and found no substantial difference (Bae et al., 2015). The American Academy of Pediatrics (AAP) recently put forth a policy statement asserting that “the reduction of toxic stress in young children ought to be a high priority for medicine as a whole” given that childhood toxic stress is associated with unhealthy lifestyles later in life, such as substance abuse, suboptimal nutrition habits, and poor physical activity (Garner et al., 2011). The AAP has argued in favor of an Ecobiodevelopmental Framework in the fight against pediatric toxic stress. The main tenants of this framework are: Oluwadamilola Oni-Orisan 17 1)! early childhood stress is a critical factor that can reduce a child’s ability to adapt and cope with challenges later in life 2)! the root causes of unhealthy lifestyles, low socioeconomic status, poor coping skills, and weak social networks lie in the behavioral and physiological responses to adversity in childhood, and 3)! stable, responsive relationships that make children feel safe yield the best defense mechanism against long-term adverse outcomes from such stress. Conclusion Little is known about the association between prenatal methamphetamine exposure and potential epigenetic modifications in the fetus. Given that toxic stress and early adversity have been identified as potential drivers of health disparities (Shonkoff et al., 2011), and findings from the Adverse Childhood Experiences Study that provide evidence for parental substance abuse as a stressor that can yield toxic stress in children (Felitti et al., 1998), it is important to establish whether or not prenatal methamphetamine exposure and toxic stress yield long-term effects on children’s neurobehavior. The literature shows that neurobehavior can be assessed using cortisone measurements. With the growing epidemic of methamphetamine use among pregnant women in the United States and the documented adverse outcomes on children born with such exposure, it is imperative that researchers gain an understanding of the pathophysiology of methamphetamine in relation to children’s neurobehavioral outcomes in early childhood. Oluwadamilola Oni-Orisan 18 References Abar, B., LaGasse, L. L., Derauf, C., Newman, E., Shah, R., Smith, L. M., Arria, A., Huestis, M., Della Grotta, S., Dansereau, L. M., Neal, C., & Lester, B. M. (2012, October 15). Examining the Relationships Between Prenatal Methamphetamine Exposure, Early Adversity, and Child Neurobehavioral Disinhibition. Psychology of Addictive Behaviors. Advance online publication. Bae YJ, Stadelmann S, Klein AM, Jaeger S, Hiemisch A, Kiess W, Ceglarek U, Gaudl A, Schaab M, von Klitzing K, Thiery J, Kratzsch J, Döhnert M. The hyporeactivity of salivary cortisol at stress test (TSST-C) in children with internalizing or externalizing disorders is contrastively associated with !-amylase. J Psychiatr Res. 2015 Dec; 71:78-88. Bale TL, Vale WW. CRF and CRF receptors: role in stress responsivity and other behaviors. Annual Review of Pharmacology and Toxicology. 2004; 44:525-57. Berman S, O’Neill J, Fears S, Bartzokis G, London ED. Abuse of Amphetamines and Structural Abnormalities in Brain. Ann NY Acad Sci. 2008 Oct; 1141: 195-220. Binder EB. 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Epigenetics of Neurobehavior in Children with Prenatal Methamphetamine Exposure ABSTRACT Objective: To assess the association between prenatal methamphetamine exposure (PME) and methylation of the gene 11-Beta hydroxysteroid dehydrogenase type 2 (11B-HSD2) Methods: The Infant Development, Environment, and Lifestyle Study (IDEAL), a prospective, longitudinal study of PME and child outcomes enrolled postpartum mother-infant dyads in California, Hawaii, Iowa, and Oklahoma. Prenatal exposure was defined by maternal self-report and/or meconium toxicology screening. Exposed and comparison groups were matched on race, birth weight, health insurance, and education in the original study. In this study at ages 10-11 years, 100 dyads from California and Hawaii were assessed for drug exposure and methylation of the gene 11B-HSD2. Hierarchical linear models were used to determine the association between PME and methylation of 11B-HSD2 with adjustment for early childhood adversity and cortisone level. Results: Methamphetamine (MA) exposure and early childhood adversity were statistically significant predictors of methylation of 11B-HSD2 at the CpG2 site. Conclusion: This is the first evidence of MA exposure as a stressor on child neurobehavior, which confirms the third pathophysiology of MA, and the first report of the epigenetic stress model in the context of MA use. Oluwadamilola Oni-Orisan 27 INTRODUCTION Methamphetamine use in the United States is a major public health problem that continues to grow.1 Of particular concern is methamphetamine use among women who are pregnant. In 2014, 0.1% of pregnant women used methamphetamine.2 In 2015, 4.7% of pregnant women aged 15-44 years used illicit drugs including methamphetamine.1 Methamphetamine is classified as a stimulant and a neurotoxin that is known to act on both the central nervous system and the sympathetic nervous system.3 While methamphetamine use is associated with a host of health risks in the general population of adult users,4,5,6,7 pregnant women who use methamphetamines are a particularly important subset of the population to study given the risk of associated developmental outcomes in their children.8 Infants born with prenatal methamphetamine exposure often develop neurobehavioral disinhibition (ND),8,9 including cognitive and behavioral deficits,10,11 and altered brain architecture, and imbalanced neurochemistry.12 These effects have been shown to extend into memory,13,14,15 attention,13 inhibitory control,16 motor control,17,18 and vision.17 The results of a 2010 study found that children with prenatal methamphetamine and alcohol exposure had significantly smaller brains than children who had been exposed to alcohol only, methamphetamine only, or neither toxin, indicating that prenatal methamphetamine exposure could be unique from other teratogens in terms of the danger that it poses to the developing fetus.12 While prenatal methamphetamine exposure (PME) is related to developmental deficits later in life, there are no studies on PME and DNA methylation in relation to long-term cognitive and behavioral deficits. Little is known about the relationship between prenatal methamphetamine exposure and epigenetic changes in the neurobehavior of the developing fetus. Epigenetic changes occur in the developing embryo via fetal programming, behavioral intervention, or germline transmission.19,20 Oluwadamilola Oni-Orisan 28 Epigenetics is essential to directing normal development and promoting the stability of the genome, particularly during sensitive periods.21 For example, epigenetic changes drive the process of cell differentiation.21 The gene 11-Beta-hydroxysteroid dehydrogenase Type II (11B-HSD2) was selected as the candidate gene for this study given its role as a stress-responsive gene that codes for an enzymatic protein. 11B-HSD2 encodes the enzymatic protein that regulates the amount of active cortisol in the blood by a mechanism that deactivates cortisol;22 deactivated cortisol is referred to as cortisone.23 During pregnancy, the enzyme protects the fetus from the mother’s cortisol by converting it into inactive cortisone.24 Methylation of 11B-HSD2 at the promoter region has been shown to alter 11B-HSD2 gene expression.25 This faulty enzymatic function results in adverse effects such as prolonged HPA reactivity, low birth weight, and increased anxiety, hyperglycemia, and hypertension in rodent models.26 Cortisone is a useful biomarker for measuring current stress.27 Salivary cortisone has been reported to accurately reflect free serum cortisol during the stimulation of the adrenal glands.28 In a study comparing the response patterns of cortisol and cortisone, researchers found that the two biomarkers exhibited a similar circadian rhythm and found no substantial difference.29 Thus, cortisone was used as a surrogate measure of current stress in this study. Many studies support the theory that early adversity also affects development by controlling HPA axis reactivity.30,31,32,33,34,35 The physiological effects of early life stress are important in studying PME because PME is associated with increased early adversity experienced by the child.8 An embryo’s exposure to prenatal stress is especially profound when it occurs during a “critical period” in fetal development, which is a window of time in which stress causes irreversible epigenetic changes.36 Research has shown that DNA methylation and Oluwadamilola Oni-Orisan 29 the critical period during which it occurs are associated with developmental outcome;37,38 when prenatal stress takes place in a certain critical period, epigenetic changes can continue to shape the embryo’s development.19 The study of human behavior through the lens of epigenetics is novel given the relatively recent development of epigenetic behavioral research. The application of epigenetics to behavioral research is of great significance since there is vast potential to use epigenetics to ascertain the molecular processes that act on gene expression and how these processes shape behavior and development in children. Of great interest is understanding how prenatal methamphetamine exposure is potentially associated with epigenetic modification of the fetus’s neuroendocrine system. This study aims to better understand the relationship between epigenetics, PME, early adversity, and stress. We hypothesized that prenatal methamphetamine exposure would be significantly associated with increased methylation. METHODS IDEAL Study Data come from the Infant Development, Environment, and Lifestyle (IDEAL) Study (for more information on IDEAL, see Smith et al., 2006, 2008).39,40 In Phase I of IDEAL, mother- child dyads were recruited from hospitals in Los Angeles, California; Honolulu, Hawaii; Des Moines, Iowa; and Tulsa, Oklahoma; areas known to have high prevalence of MA use. Mothers were recruited for the study between delivery and discharge. Procedures at all sites were approved by the Institutional Review Board. All participants reviewed and signed a written informed consent form. Oluwadamilola Oni-Orisan 30 The IDEAL study screened 34,883 mother-child dyads upon delivery. Of these, 26,999 mothers were screened for eligibility. Mothers were excluded using the following exclusion criteria: (1) Non-English speaking (17.7%), (2) severe psychological impairment (0.1%), (3) low cognitive functioning (0.2%), (4) younger than 18 years of age (3.5%), and (5) the use of cocaine, opiates, LSD/hallucinogens, or PCP (2.2%). Exclusion criteria for infants were: (1) multiple birth (4.5%), (2) congenital or chromosomal anomalies (0.5%), (3) unlikely to survive (0.5%), and (4) overt TORCH (Toxoplasmosis, other agents, rubella, cytomegalovirus, herpes simplex) infection (0.07%). Upon further screening, 17,961 were deemed eligible for participation. Of those, 3,705 (21%) provided consent to participate in enrollment. Only mothers with prenatal MA use (n=204) and their matched controls (n=208) were enrolled in the IDEAL study (n=412). Mothers in the control group were matched to those in the MA exposure group by child’s birthweight, maternal race, maternal education, and type of insurance. The project obtained a National Institute on Drug Abuse Certificate of Confidentiality to ensure confidentiality and truthfulness in reporting maternal drug use. Following the initial in- hospital visit upon the child’s birth, follow-up visits were conducted when the child was 1 month, 12 months, 24 months, 36 months, 5 years, 5.5 years, 6.5 years, 7 years, 7.5 years, and 10-11 years of age. Participants in Current Study The current study uses dyads recruited from California and Hawaii who completed the 10-11 year follow-up visit (n=100). These 100 mother-child pairs were categorized by MA exposed (n=55) and comparison (n=45). Oluwadamilola Oni-Orisan 31 Measures and Procedures PME and other Substance Use. PME was determined via (a) mothers’ self-reported MA use and/or (b) confirmation of methamphetamine metabolites in infant meconium using gas chromatography-mass spectrometry (GCMS) and positive immunoassay. The meconium of each infant was screened. Upon a presumptive positive, GCMS was performed in order to identify the specific metabolites that were present. Tobacco, alcohol, and marijuana use during pregnancy were assessed via maternal self-report. Early Adversity. Early adversity was coded using a single index score similar to those used in previous studies.41,42 The early adversity index accounted for cumulative measures of adversity from birth through age 5 years by calculating the sum of a set of binary indicators (Table 1). Cortisone. A proximal crown head hair sample was obtained from the child at the 10-11 year study visit. The 3-cm hair segment was analyzed by the United States Drug Testing Laboratories (USDTL) using a liquid chromatography-tandem mass spectrometry assay. Specimens were analyzed according to a slightly modified version of a previously published procedure.43 Hair data was collected for 89 subjects (5 refused, 1 hair too short, 1 religion, 4 no reason provided). Cortisone was used to measure current stress. DNA Methylation Analysis. A sodium bisulfite pyrosequencing approach was used to assess the methylation status of 11B-HSD2 CpG sites 1-4. Primers were taken from the literature (112) or designed using regions described in the literature (115, 116, 184). PSQ Assay Design software (Qiagen, Inc) was used to amplify the sodium bisulfite modified DNA for each CpG site. The performance characteristics of the assay were assessed using dilution series of fully methylated referent DNA into fully unmethylated referent DNA. The PyroMark MD system was Oluwadamilola Oni-Orisan 32 used for all pyrosequencing assays. Quantitative assessment of the extent of DNA methylation at each CpG site was performed by using the integrated Pyro-Q-CpG software (Qiagen) to analyze data. Statistical Analysis One-way analysis of variance (ANOVA) was used for analysis of continuous measures and Chi-square for categorical measures. Hierarchical multiple regression using SPSS (version 24.0.0.0) examined the effects of prenatal MA exposure, early life stress, and current life stress on methylation of 11B-HSD2 at four CpG sites after controlling for covariates. Methylation variables for the CpG1, CpG2, CpG3, and CpG4 sites were winsorized and natural log transformed. The models were adjusted for the following covariates: gestational age at birth, quantity of self-reported use of tobacco, alcohol, and marijuana use. Covariates were selected based on maternal and child characteristics that significantly differed between mothers in the MA exposure and comparison groups (P<0.05) yet were not highly correlated with other covariates (r>0.70), trends in the literature, and conceptual reasons. RESULTS There were no differences in demographic characteristics between children who were included in the study and those who were not (Table 2). Included mothers reported greater prenatal alcohol use and differed by race (Table 2). Maternal and child characteristics were also compared across MA exposure (Table 3). PME children were exposed to significantly more tobacco and marijuana in utero, were more likely to be born to single mothers, and less likely to have received prenatal care. At birth, PME infants weighed on average 267 g less, measured 2.9 cm shorter, had 0.7 cm smaller head Oluwadamilola Oni-Orisan 33 circumference, and were 1.36 weeks younger than comparison children. PME mothers reported low socioeconomic status. To determine if PME was associated with methylation of 11B-HSD2 at any of the four CpG sites, the initial model looked at methylation as the dependent variable and PME as the only predictor variable (Table 4). CpG2 was the only methylation site associated with PME. In model 1, PME children had higher levels of DNA methylation at the CpG2 site than comparison children (P=0.004) (Table 4). Early adversity was added as a predictor in the second CpG2 model. A higher score on the early adversity index was also related to higher levels of DNA methylation at the CpG2 site (P=0.011), but PME was no longer significant. In the full model (Model 4), covariates were added, including: gestational age at birth, and count variables reflecting the quantity of tobacco, alcohol, and marijuana consumed prenatally. None of the covariates were related to DNA methylation at the CpG2 site, but the effects of adversity (P=0.014) and PME (P=0.025) on DNA methylation at this site remained statistically significant. DISCUSSION This is the first study to measure the effects of PME on a stress-related gene. It is also the first study to measure the effects of early adversity on a stress-related gene. We found that PME was associated with methylation of 11B-HSD2 at CpG site 2. After adjusting for covariates, both PME and early adversity are associated with increased methylation. These findings are the first of their kind. This study shows long-term effects of PME in children. It also shows that methylation affected by both PME and early adversity has long-term effects. Children with high PME have been found to have increased cortisol reactivity, a relationship modified by the Oluwadamilola Oni-Orisan 34 experience of mild to moderate potential for child physical abuse.44 Increased cortisol reactivity has also been noted in children45 and adolescents46 who were prenatally exposed to cocaine.47 Studies have also shown that early life stress (early adversity) can result in either increased or decreased cortisol reactivity, a relationship modified by chronicity and severity.48,49 Researchers have proposed that early adversity can induce irreversible epigenetic effects on certain genes that may increase a child’s susceptibility to neuroendocrine and behavioral dysfunction.50,51,52 An early IDEAL study examined the relationship between PME, early adversity, and neurobehavioral disinhibition and found that high levels of early adversity were associated with greater behavioral and emotional control problems at age 5 years.8 The present study is the first to evaluate PME, early adversity, and the epigenetics of neurobehavior at age 10-11 years. The rationale for studying 11B-HSD2, a glucocorticoid stress-related gene, was that it permitted the study of methamphetamine as a stressor and a modifier of the expression of associated genes. This permitted us to test the hypothesis of the third pathophysiology (epigenetic stress model) and also ask whether or not the pathway could be generalized beyond prenatal cocaine exposure. The results of this study support the hypothesis and the generalizability of this model. In studying the epigenetics of prenatal cocaine exposure, Lester and Padbury summarized the pathophysiology of cocaine along two known pathways (neurochemical and vasoconstrictive), and proposed a third pathophysiological pathway: the epigenetic stress model.53 Their hypothetical model postulated that cocaine, in addition to acting as a neurotransmitter blocker in the neurochemical pathway and promoter of plasma catecholamine concentrations in the vasoconstrictive pathway, could also act as a stressor that disrupts neuroendocrine homeostasis and normal fetal programming.53 Such disruptions in the prenatal environment would result in decreased levels of key placental genes that program the Oluwadamilola Oni-Orisan 35 fetal neuroendocrine environment in utero.53 This could also apply to other drugs and stressors. This study is the first to yield evidence that supports the validity of this model. We assessed DNA methylation on 11B-HSD2’s promoter region because this region regulates the neuroendocrine system, including the HPA axis and stress response.54 Research has shown that DNA methylation at CpG sites in the promoter and first exon of 11B-HSD2 can modify the expression of this gene.55 Increased methylation inhibits the binding of transcription factors to binding sites on the promoter region; the four transcription factors of interest concerning 11B-HSD2 are E2F1, GR-!, nuclear factor 1 (NF-1), and specificity protein 1 (Sp1). In silico analyses indicate that CpG2 lies in a sequence region that also contains binding sites for the E2F1 transcription factor, which regulates DNA synthesis, cell cycle regulation, cell proliferation, and apoptosis.54 A GR-! binding region for GR-!, which regulates 11B-HSD2 expression via a negative feedback loop, is also adjacent to CpG2.54 This implies that methylation in the promoter region affects 11B-HSD2 expression by inhibiting binding of E2F1 or GR.54 Furthermore, the transcriptional activity of 11B-HSD2 is reduced when increased methylation inhibits the binding of NF-1 (4 binding sites) and Sp1 (11 binding sites).55 Sp1 codes for a protein that regulates several cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling.56 The NF-1 family of nuclear factors encodes proteins that control vital processes in central nervous system development including axon guidance and outgrowth, glial and neuronal cell differentiation, and neuronal migration.57 These proteins also control the formation of midline glia in the cortex58 and gliogenesis in the spinal cord.59 Thus, the inhibition of NF-1 binding could yield abnormal brain development, particularly in midline structures, and spinal cord abnormalities that cause Oluwadamilola Oni-Orisan 36 neurogenic urinary tract defects.60 Modified expression of NF-1 genes may also produce clinical presentations similar to callosal agenesis and macrocephaly.60 PME and early adversity both affect stress related genes by methylating key binding sites, which implies that both exposures could inhibit the binding of key transcription factors, resulting in decreased gene expression. Methylation of stress-related genes is disruptive because it inhibits the ability of the glucocorticoid receptors that they encode to properly bind glucocorticoids such as cortisol in humans.61 As a result, the negative feedback mechanism that normally regulates active cortisol is modified; instead of binding active cortisol and stopping further production of it, active cortisol remains in the bloodstream. Thus, the brain never receives the signal that it needs to reduce cortisol production. The HPA axis remains activated. This prolonged activation of the stress response system can result in toxic stress, the most dangerous stress response.62 Toxic stress is dangerous because it is believed to disrupt brain circuitry normal organ and metabolic systems during sensitive development periods, which can produce physiological changes that lead to learning and behavior impairments, and physical and mental illness.62 There are limitations to these findings. Experimental work was limited given that tissue such as brain matter cannot be collected from living participants. Samples were collected from peripheral tissue, however it is unknown whether or not peripheral tissue such as placenta, cord blood, and buccal cells experience identical epigenetic changes.19 Epigenetic mechanisms are responsible for cellular differentiation and tissue specificity, therefore it is unlikely that patterns in DNA methylation in buccal cells will exactly match those of neural tissue. Nonetheless, buccal cells and neural cells are both derived from the same germ layer, the endoderm, during fetal development. Secondly, there was a notable absence of cortisol in the hair. Raul et al. measured cortisone and cortisol in human hair and found that cortisone concentrations were Oluwadamilola Oni-Orisan 37 higher than cortisol concentrations, unlike plasma in which concentrations of cortisol are significantly higher than that of cortisone.43 It was postulated that this might be due to increased activity of 11B-HSD2 (the enzyme responsible for converting cortisol to inactive cortisone) in the hair bulb.63 Conclusion This is the first test of Padbury and Lester’s third pathophysiology of prenatal drug exposure. These findings provide the first insight into the relationship between the epigenetics of neurobehavior in children with PME. Before this study, there was no evidence that supported the potential association between prenatal methamphetamine exposure and epigenetic modifications in the fetus. Given the growing epidemic of methamphetamine use among pregnant women in the United States and the documented developmental outcomes of children born with such exposure, it is imperative that researchers gain an understanding of the pathophysiology of methamphetamine in relation to children’s neurobehavioral outcomes in early childhood. Studies have shown that epigenetic changes can alter normal child development and behavior.64-68,54,69 For example, researchers have found that prenatal and postnatal programming regulates epigenetic modifications in children to adjust to environmental adversity (Lester Conradt 2014). That study found that infants at the highest risk for depression experienced maternal depression both pre- and post-partum, periods in which adversity could further aggravate depression. Fetal exposure to maternal depression during pregnancy results in elevated circulating cortisol, elevated norepinephrine, and decreased serotonin. The placental genes that regulate uptake of serotonin (SLC6A4) and control the neuroendocrine environment (NET, 11B- HSD2, NR3C1) reprogram the HPA axis, thereby modifying postnatal baseline levels of Oluwadamilola Oni-Orisan 38 performance of the HPA axis and related physiological systems. This reprogramming results in increased cortisol secretion, decreased serotonin secretion, and thus a lowered threshold of HPA reactivity to the postnatal environment in anticipation of further exposure to adversity in the postnatal environment. After birth, the increased physiological reactivity (cortisol secretion) is protective against early adversity in the short-term, but makes the child more susceptible to long term chronic stress (toxic stress) and the effects of maternal postpartum depression.67 Toxic stress and early adversity are potential drivers of health disparities.62 The Adverse Childhood Experiences Study found that parental substance abuse is a stressor that can yield toxic stress in children.71 Thus, in working to reduce health disparities, researchers and clinicians must recognize that PME puts children at risk for long-term vulnerabilities. Oluwadamilola Oni-Orisan 39 Table 1. The early adversity index accounted for cumulative measures of adversity from birth through age 5 years by calculating the sum of a set of binary indicators Binary indicators of adversity any self-reported maternal postnatal substance use through 3 years any extreme poverty experienced between birth and 5 years (as indicated by annual income <$10,000, approximately 50% of the U.S. Department of Health and Human Services poverty line for families with two to five members at the time of data collection) any changes in the primary caregiver through 5 years any maternal subscale score on the Brief Symptom Inventory greater than the clinical cut-off point (Derogatis, 1993) through 3 years maternal depression at least one standard deviation above the mean from birth through 3 years indicated by the Beck Depression Inventory (M=9.44, SD=7.03; Beck, Steer & Brown, 1996) quality of the living environment at least one standard deviation below the mean at 2.5 years as indicated by the Home Inventory (M=37.69, SD=4.39; Caldwell & Bradley, 2001) community violence at least one standard deviation above the mean from birth through 3 years as indicated by the Neighborhood Problems section of the Lifestyle Interview (M=1.70, SD=1.84) social position at least one standard deviation below the sample mean from birth through 5 years as indicated by the Index of Social Position which reflects a weighted average of parental occupational status and education level (M=31.32, SD=8.92; see Hollingshead, 1975; LaGasse et al., 1999) Oluwadamilola Oni-Orisan 40 Table 2. Comparison of dyads included and not included N (%) or mean (SD) Included Excluded p-value N=100 N=212 Maternal/demographic characteristics Race 0.008 •! White 33 (33.0%) 49 (23.1%) •! Hispanic 35 (35.0%) 51 (24.1%) •! Hawaii & Pacific Islander 15 (15.0%) 56 (26.4%) •! Asian 12 (12.0%) 45 (21.2%) •! Black 3 (3.0%) 11 (5.2%) •! American Indian 1 (1.0%) 0 (0.0%) •! Other 1 (1.0%) 0 (0.0%) Low SES 27 (27%) 59 (28.1%) 0.840 Partner at birth 53 (53.0%) 118 (55.7%) 0.659 Education <12 years 48 (48.0%) 95 (45.0%) 0.623 Prenatal care 95 (95.0%) 196 (92.5%) 0.402 Prenatal tobacco use 50 (50.0%) 115 (54.2%) 0.483 Average number of cigarettes/day 3.20 (5.87) 4.20 (7.40) 0.240 across pregnancy Prenatal alcohol use 30 (30.0%) 41 (19.3%) 0.036 Average oz. absolute alcohol/day 0.05 (0.30) 0.07 (0.43) 0.708 across pregnancy Prenatal marijuana use 17 (17.0%) 31 (14.6%) 0.587 Average number of joints/day 0.03 (0.15) 0.09 (1.03) 0.528 across pregnancy Prenatal methamphetamine (MA) 55 (55.0%) 102 (48.1%) 0.256 use Maternal age (yr) 25.24 (6.21) 25.38 (5.78) 0.849 Neonatal characteristics Sex (Male) 52 (52.0%) 115 (54.2%) 0.711 Birth weight (g) 3229 (642) 3234 (580) 0.946 Length (cm) 50.0 (3.9) 51 (3.0) 0.168 Head Circumference (cm) 33.8 (1.9) 33.9 (1.8) 0.766 Gestational age (wk) 38.4 (2.6) 38.7 (1.8) 0.253 Small for gestational age 10 (10.0%) 30 (14.2%) 0.306 Oluwadamilola Oni-Orisan 41 Table 3. Maternal and neonatal characteristics by MA exposure N (%) or mean (SD) MA Exposed Comparison p-value N=55 N=45 Maternal/demographic characteristics Race 0.721 •! White 18 (32.7%) 15 (33.3%) •! Hispanic 18 (32.7%) 17 (37.8%) •! Hawaii & Pacific Islander 10 (18.2%) 5 (11.1%) •! Asian 7 (12.7%) 5 (11.1%) •! Black 1 (1.8%) 2 (4.4%) •! American Indian 1 (1.8%) 0 (0.0%) •! Other 0 (0.0%) 1 (2.2%) Low SES 23 (41.8%) 4 (8.9%) <0.001 Partner at birth 24 (43.6%) 29 (64.4%) 0.038 Education <12 years 31 (56.4%) 17 (37.8%) 0.064 Prenatal care 50 (90.9%) 45 (100.0%) 0.038 Prenatal tobacco use 40 (72.7%) 10 (22.2%) <0.001 Average number of cigarettes/day 5.50 (7.06) 0.44 (1.50) <0.001 across pregnancy Prenatal alcohol use 18 (32.7%) 12 (26.7%) 0.511 Average oz. absolute alcohol/day 0.09 (0.40) 0.01 (0.03) 0.177 across pregnancy Prenatal marijuana use 15 (27.3%) 2 (4.4%) 0.002 Average number of joints/day 0.03 (0.14) 0.02 (0.15) 0.754 across pregnancy Maternal age (yr) 25.11 (5.79) 25.40 (6.75) 0.817 GA at 1st prenatal visit 13.6 (8.04) 8.2 (5.77) <0.001 Neonatal characteristics Sex (Male) 31 (56.4%) 21 (46.7%) 0.334 Birth weight (g) 3109 (687) 3376 (554) 0.037 Length (cm) 48.7 (4.1) 51.6 (2.8) <0.001 Head Circumference (cm) 33.5 (2.0) 34.2 (1.6) 0.047 Gestational age (wk) 37.75 (3.12) 39.11 (1.60) 0.009 Small for gestational age 6 (10.9%) 4 (8.9%) 0.738 Oluwadamilola Oni-Orisan 42 Table 4. Summary of multivariate analysis for predicting methylation at CpG2 of 11B- HSD2 Model 1 Model 2 Model 3 Model 4 Variable Estimate SE P Estimate SE P Estimate SE p Estimate SE p Step 1 PM Exposure 0.34 0.12 0.004 0.23 0.12 0.063 0.24 0.12 0.048 0.32 0.14 0.025 Step 2 Early adversity 0.11 0.04 0.011 0.11 0.04 0.010 0.11 0.04 0.014 Step 3 -0.13 0.11 0.232 -0.13 0.12 0.246 Cortisone Step 4 Avg # of cigs/day -0.01 0.01 0.574 Avg absolute -0.21 0.20 0.299 alcohol oz./day Avg # joints/day 0.39 0.40 0.337 Gestational age 0.02 0.02 0.377 Oluwadamilola Oni-Orisan 43 References 1.! Center for Behavioral Health Statistics and Quality. (2016). 2015 National Survey on Drug Use and Health: Detailed Tables. Substance Abuse and Mental Health Services Administration, Rockville, MD. 2.! Center for Behavioral Health Statistics and Quality. (2015). 2014 National Survey on Drug Use and Health: Detailed Tables. Substance Abuse and Mental Health Services Administration, Rockville, MD. 3.! 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