DNA methylation of imprint control regions associated with Alzheimer's disease in non-Hispanic Blacks and non-Hispanic Whites.

Alzheimer's disease (AD) prevalence is twice as high in non-Hispanic Blacks (NHBs) as in non-Hispanic Whites (NHWs). The objective of this study was to determine whether aberrant methylation at imprint control regions (ICRs) is associated with AD. Differentially methylated regions (DMRs) were bioinformatically identified from whole-genome bisulfite sequenced DNA derived from brain tissue of 9 AD (5 NHBs and 4 NHWs) and 8 controls (4 NHBs and 4 NHWs). We identified DMRs located within 120 regions defined as candidate ICRs in the human imprintome ( https://genome.ucsc.edu/s/imprintome/hg38.AD.Brain_track ). Eighty-one ICRs were differentially methylated in NHB-AD, and 27 ICRs were differentially methylated in NHW-AD, with two regions common to both populations that are proximal to the inflammasome gene, NLRP1, and a known imprinted gene, MEST/MESTIT1. These findings indicate that early developmental alterations in DNA methylation of regions regulating genomic imprinting may contribute to AD risk and that this epigenetic risk differs between NHBs and NHWs.

Creation and Validation of the First Infinium DNA Methylation Array for the Human Imprintome.

Background: Differentially methylated imprint control regions (ICRs) regulate the monoallelic expression of imprinted genes. Their epigenetic dysregulation by environmental exposures throughout life results in the formation of common chronic diseases. Unfortunately, existing Infinium methylation arrays lack the ability to profile these regions adequately. Whole genome bisulfite sequencing (WGBS) is the unique method able to profile these regions, but it is very expensive and it requires not only a high coverage but it is also computationally intensive to assess those regions. Findings: To address this deficiency, we developed a custom methylation array containing 22,819 probes. Among them, 9,757 probes map to 1,088 out of the 1,488 candidate ICRs recently described. To assess the performance of the array, we created matched samples processed with the Human Imprintome array and WGBS, which is the current standard method for assessing the methylation of the Human Imprintome. We compared the methylation levels from the shared CpG sites and obtained a mean R 2 = 0.569. We also created matched samples processed with the Human Imprintome array and the Infinium Methylation EPIC v2 array and obtained a mean R 2 = 0.796. Furthermore, replication experiments demonstrated high reliability (ICC: 0.799-0.945). Conclusions: Our custom array will be useful for replicable and accurate assessment, mechanistic insight, and targeted investigation of ICRs. This tool should accelerate the discovery of ICRs associated with a wide range of diseases and exposures, and advance our understanding of genomic imprinting and its relevance in development and disease formation throughout the life course.

Genomic map of candidate human imprint control regions: the imprintome.

Imprinted genes - critical for growth, metabolism, and neuronal function - are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

The science of hope: an interview with Randy Jirtle.

In this interview, Professor Randy L Jirtle speaks with Storm Johnson, Commissioning Editor for Epigenomics, on his work on genomic imprinting, environmental epigenomics and the fetal origins of disease susceptibility. Professor Randy Jirtle joined the Duke University Department of Radiology in 1977 and headed the Epigenetics and Imprinting Laboratory until 2012. He is now Professor of Epigenetics in the Department of Biological Sciences at North Carolina State University, Raleigh, NC, USA. Jirtle's research interests are in epigenetics, genomic imprinting and the fetal origins of disease susceptibility. He is known for his groundbreaking studies linking environmental exposures early in life to the development of adult diseases through changes in the epigenome and for determining the evolutionary origin of genomic imprinting in mammals. He has published over 200 peer-reviewed articles as well as the books Liver Regeneration andCarcinogenesis: Molecular and Cellular Mechanisms, Environmental Epigenomics in Health and Disease: Epigenetics and Disease Origins and Environmental Epigenomics in Health andDisease: Epigenetics and Complex Diseases. He was honored in 2006 with the Distinguished Achievement Award from the College of Engineering at the University of Wisconsin-Madison. In 2007, he was a featured scientist on the NOVA television program on epigenetics titled 'Ghost in Your Genes' and was nominated for Time Magazine's 'Person of the Year'. He was the inaugural recipient of the Epigenetic Medicine Award in 2008 and received the STARS Lecture Award in Nutrition and Cancer from the National Cancer Institute in 2009. Jirtle was presented the Linus Pauling Award from the Institute of Functional Medicine in 2014. In 2017, ShortCutsTV produced the English documentary 'Are You What Your Mother Ate? The Agouti Mouse Study' based on his pioneering epigenetic research. He received the 2018 Northern Communities Health Foundation Visiting Professorship Award at the University of Adelaide, Australia. The Personalized Lifestyle Medicine Institute presented Jirtle with the Research and Innovation Leadership Award in 2019. Dr Jirtle was also given the Alexander Hollaender Award in 2019 at the 50th annual meeting of the Environmental Mutagenesis and Genomics Society.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Cadmium exposure and MEG3 methylation differences between Whites and African Americans in the NEST Cohort.

Cadmium (Cd) is a ubiquitous environmental pollutant associated with a wide range of health outcomes including cancer. However, obscure exposure sources often hinder prevention efforts. Further, although epigenetic mechanisms are suspected to link these associations, gene sequence regions targeted by Cd are unclear. Aberrant methylation of a differentially methylated region (DMR) on the MEG3 gene that regulates the expression of a cluster of genes including MEG3, DLK1, MEG8, MEG9 and DIO3 has been associated with multiple cancers. In 287 infant-mother pairs, we used a combination of linear regression and the Getis-Ord Gi* statistic to determine if maternal blood Cd concentrations were associated with offspring CpG methylation of the sequence region regulating a cluster of imprinted genes including MEG3. Correlations were used to examine potential sources and routes. We observed a significant geographic co-clustering of elevated prenatal Cd levels and MEG3 DMR hypermethylation in cord blood (P = 0.01), and these findings were substantiated in our statistical models (ß = 1.70, se = 0.80, P = 0.03). These associations were strongest in those born to African American women (ß = 3.52, se = 1.32, P = 0.01) compared with those born to White women (ß = 1.24, se = 2.11, P = 0.56) or Hispanic women (ß = 1.18, se = 1.24, P = 0.34). Consistent with Cd bioaccumulation during the life course, blood Cd levels increased with age (ß = 0.015 µg/dl/year, P = 0.003), and Cd concentrations were significantly correlated between blood and urine (ρ > 0.47, P < 0.01), but not hand wipe, soil or house dust concentrations (P > 0.05). Together, these data support that prenatal Cd exposure is associated with aberrant methylation of the imprint regulatory element for the MEG3 gene cluster at birth. However, neither house-dust nor water are likely exposure sources, and ingestion via contaminated hands is also unlikely to be a significant exposure route in this population. Larger studies are required to identify routes and sources of exposure.

Enhancing and Extending Biological Performance and Resilience.

Human performance, endurance, and resilience have biological limits that are genetically and epigenetically predetermined but perhaps not yet optimized. There are few systematic, rigorous studies on how to raise these limits and reach the true maxima. Achieving this goal might accelerate translation of the theoretical concepts of conditioning, hormesis, and stress adaptation into technological advancements. In 2017, an Air Force-sponsored conference was held at the University of Massachusetts for discipline experts to display data showing that the amplitude and duration of biological performance might be magnified and to discuss whether there might be harmful consequences of exceeding typical maxima. The charge of the workshop was "to examine and discuss and, if possible, recommend approaches to control and exploit endogenous defense mechanisms to enhance the structure and function of biological tissues." The goal of this white paper is to fulfill and extend this workshop charge. First, a few of the established methods to exploit endogenous defense mechanisms are described, based on workshop presentations. Next, the white paper accomplishes the following goals to provide: (1) synthesis and critical analysis of concepts across some of the published work on endogenous defenses, (2) generation of new ideas on augmenting biological performance and resilience, and (3) specific recommendations for researchers to not only examine a wider range of stimulus doses but to also systematically modify the temporal dimension in stimulus inputs (timing, number, frequency, and duration of exposures) and in measurement outputs (interval until assay end point, and lifespan). Thus, a path forward is proposed for researchers hoping to optimize protocols that support human health and longevity, whether in civilians, soldiers, athletes, or the elderly patients. The long-term goal of these specific recommendations is to accelerate the discovery of practical methods to conquer what were once considered intractable constraints on performance maxima.

DNA methylation of imprinted gene control regions in the regression of low-grade cervical lesions.

The role of host epigenetic mechanisms in the natural history of low-grade cervical intraepithelial neoplasia (CIN1) is not well characterized. We explored differential methylation of imprinted gene regulatory regions as predictors of the risk of CIN1 regression. A total of 164 patients with CIN1 were recruited from 10 Duke University clinics for the CIN Cohort Study. Participants had colposcopies at enrollment and up to five follow-up visits over 3 years. DNA was extracted from exfoliated cervical cells for methylation quantitation at CpG (cytosine-phosphate-guanine) sites and human papillomavirus (HPV) genotyping. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression to quantify the effect of methylation on CIN1 regression over two consecutive visits, compared to non-regression (persistent CIN1; progression to CIN2+; or CIN1 regression at a single time-point), adjusting for age, race, high-risk HPV (hrHPV), parity, oral contraceptive and smoking status. Median participant age was 26.6 years (range: 21.0-64.4 years), 39% were African-American, and 11% were current smokers. Most participants were hrHPV-positive at enrollment (80.5%). Over one-third of cases regressed (n = 53, 35.1%). Median time-to-regression was 12.6 months (range: 4.5-24.0 months). Probability of CIN1 regression was negatively correlated with methylation at IGF2AS CpG 5 (HR = 0.41; 95% CI = 0.23-0.77) and PEG10 DMR (HR = 0.80; 95% CI = 0.65-0.98). Altered methylation of imprinted IGF2AS and PEG10 DMRs may play a role in the natural history of CIN1. If confirmed in larger studies, further research on imprinted gene DMR methylation is warranted to determine its efficacy as a biomarker for cervical cancer screening.

Non-genomic transmission of longevity between generations: potential mechanisms and evidence across species.

Accumulating animal and human data indicate that environmental exposures experienced during sensitive developmental periods may strongly influence risk of adult disease. Moreover, the effects triggered by developmental environmental cues can be transgenerationally transmitted, potentially affecting offspring health outcomes. Increasing evidence suggests a central role of epigenetic mechanisms (heritable alterations in gene expression occurring without changes in underlying DNA sequence) in mediating these effects. This review summarizes the findings from animal models, including worms, insects, and rodents, and also from human studies, indicating that lifespan and longevity-associated characteristics can be transmitted across generations via non-genetic factors.

Analysis of Imprinted Gene Regulation.

Genetic studies have been well established for identifying sequence variants associated with phenotypes. With the expanding field of epigenetics, and the growing understanding of epigenetic regulation of gene expression, similar studies can be undertaken to also define associations between epigenetic variation and phenotypes. Of particular interest are imprinted genes, which have parent-of-origin specific regulation and expression, and are key regulators of early development. Herein, we describe methods for examining epigenetic regulation by the two major hallmarks of imprinted genes: differentially methylated regions (DMRs), regulatory DNA sequences with allele specific methylation; and monoallelic expression, the silencing and transcription of opposite alleles in a parent-of-origin specific manner.

Epigenetics, obesity and early-life cadmium or lead exposure.

Obesity is a complex and multifactorial disease, which likely comprises multiple subtypes. Emerging data have linked chemical exposures to obesity. As organismal response to environmental exposures includes altered gene expression, identifying the regulatory epigenetic changes involved would be key to understanding the path from exposure to phenotype and provide new tools for exposure detection and risk assessment. In this report, we summarize published data linking early-life exposure to the heavy metals, cadmium and lead, to obesity. We also discuss potential mechanisms, as well as the need for complete coverage in epigenetic screening to fully identify alterations. The keys to understanding how metal exposure contributes to obesity are improved assessment of exposure and comprehensive establishment of epigenetic profiles that may serve as markers for exposures.

DNA Methylation of Regulatory Regions of Imprinted Genes at Birth and Its Relation to Infant Temperament.

BACKGROUND: DNA methylation of the differentially methylated regions (DMRs) of imprinted genes is relevant to neurodevelopment. METHODS: DNA methylation status of the DMRs of nine imprinted genes in umbilical cord blood leukocytes was analyzed in relation to infant behaviors and temperament (n = 158). RESULTS: MEG3 DMR levels were positively associated with internalizing (ß = 0.15, P = 0.044) and surgency (ß = 0.19, P = 0.018) behaviors, after adjusting for birth weight, gender, gestational age at birth, maternal age at delivery, race/ethnicity, education level, smoking status, parity, and a history of anxiety or depression. Higher methylation levels at the intergenic MEG3-IG methylation regions were associated with surgency (ß = 0.28, P = 0.0003) and PEG3 was positively related to externalizing (ß = 0.20, P = 0.01) and negative affectivity (ß = 0.18, P = 0.02). CONCLUSION: While the small sample size limits inference, these pilot data support gene-specific associations between epigenetic differences in regulatory regions of imprinted domains at birth and later infant temperament.

Maternal blood lead concentrations, DNA methylation of MEG3 DMR regulating the DLK1/MEG3 imprinted domain and early growth in a multiethnic cohort.

Prenatal exposure to lead (Pb) is known to decrease fetal growth; but its effects on postnatal growth and mechanistic insights linking Pb to growth are not clearly defined. Genomically imprinted genes are powerful regulators of growth and energy utilization, and may be particularly vulnerable to environmental Pb exposure. Because imprinting is established early and maintained via DNA methylation, we hypothesized that prenatal Pb exposure alters DNA methylation of imprinted genes resulting in lower birth weight and rapid growth. Pb was measured by inductively coupled plasma mass spectrometry (ICP-MS) in peripheral blood of 321 women of the Newborn Epigenetic STudy (NEST) obtained at gestation ~12 weeks. Linear and logistic regression models were used to evaluate associations between maternal Pb levels, methylation of differentially methylated regions (DMRs) regulating H19, MEG3, PEG3, and PLAGL1, measured by pyrosequencing, birth weight, and weight-for-height z score gains between birth and age 1yr, ages 1-2yrs, and 2-3yrs. Children born to women with Pb levels in the upper tertile had higher methylation of the regulatory region of the MEG3 DMR imprinted domain (ß= 1.57, se= 0.82, p= 0.06). Pb levels were also associated with lower birth weight (ß= -0.41, se= 0.15, p= 0.01) and rapid gains in adiposity (OR= 12.32, 95%CI=1.25-121.30, p= 0.03) by age 2-3 years. These data provide early human evidence for Pb associations with hypermethylation at the MEG3 DMR regulatory region and rapid adiposity gain-a risk factor for childhood obesity and cardiometabolic diseases in adulthood.

Lead Exposure during Early Human Development and DNA Methylation of Imprinted Gene Regulatory Elements in Adulthood.

BACKGROUND: Lead exposure during early development causes neurodevelopmental disorders by unknown mechanisms. Epidemiologic studies have focused recently on determining associations between lead exposure and global DNA methylation; however, such approaches preclude the identification of loci that may alter human disease risk. OBJECTIVES: The objective of this study was to determine whether maternal, postnatal, and early childhood lead exposure can alter the differentially methylated regions (DMRs) that control the monoallelic expression of imprinted genes involved in metabolism, growth, and development. METHODS: Questionnaire data and serial blood lead levels were obtained from 105 participants (64 females, 41 males) of the Cincinnati Lead Study from birth to 78 months. When participants were adults, we used Sequenom EpiTYPER assays to test peripheral blood DNA to quantify CpG methylation in peripheral blood leukocytes at DMRs of 22 human imprinted genes. Statistical analyses were conducted using linear regression. RESULTS: Mean blood lead concentration from birth to 78 months was associated with a significant decrease in PEG3 DMR methylation (ß = -0.0014; 95% CI: -0.0023, -0.0005, p = 0.002), stronger in males (ß = -0.0024; 95% CI: -0.0038, -0.0009, p = 0.003) than in females (ß = -0.0009; 95% CI: -0.0020, 0.0003, p = 0.1). Elevated mean childhood blood lead concentration was also associated with a significant decrease in IGF2/H19 (ß = -0.0013; 95% CI: -0.0023, -0.0003, p = 0.01) DMR methylation, but primarily in females, (ß = -0.0017; 95% CI: -0.0029, -0.0006, p = 0.005) rather than in males, (ß = -0.0004; 95% CI: -0.0023, 0.0015, p = 0.7). Elevated blood lead concentration during the neonatal period was associated with higher PLAGL1/HYMAI DMR methylation regardless of sex (ß = 0.0075; 95% CI: 0.0018, 0.0132, p = 0.01). The magnitude of associations between cumulative lead exposure and CpG methylation remained unaltered from 30 to 78 months. CONCLUSIONS: Our findings provide evidence that early childhood lead exposure results in sex-dependent and gene-specific DNA methylation differences in the DMRs of PEG3, IGF2/H19, and PLAGL1/HYMAI in adulthood. CITATION: Li Y, Xie C, Murphy SK, Skaar D, Nye M, Vidal AC, Cecil KM, Dietrich KN, Puga A, Jirtle RL, Hoyo C. 2016. Lead exposure during early human development and DNA methylation of imprinted gene regulatory elements in adulthood. Environ Health Perspect 124:666-673; http://dx.doi.org/10.1289/ehp.1408577.

Maternal stress, preterm birth, and DNA methylation at imprint regulatory sequences in humans.

In infants exposed to maternal stress in utero, phenotypic plasticity through epigenetic events may mechanistically explain increased risk of preterm birth (PTB), which confers increased risk for neurodevelopmental disorders, cardiovascular disease, and cancers in adulthood. We examined associations between prenatal maternal stress and PTB, evaluating the role of DNA methylation at imprint regulatory regions. We enrolled women from prenatal clinics in Durham, NC. Stress was measured in 537 women at 12 weeks of gestation using the Perceived Stress Scale. DNA methylation at differentially methylated regions (DMRs) associated with H19, IGF2, MEG3, MEST, SGCE/PEG10, PEG3, NNAT, and PLAGL1 was measured from peripheral and cord blood using bisulfite pyrosequencing in a sub-sample of 79 mother-infant pairs. We examined associations between PTB and stress and evaluated differences in DNA methylation at each DMR by stress. Maternal stress was not associated with PTB (OR = 0.98; 95% CI, 0.40-2.40; P = 0.96), after adjustment for maternal body mass index (BMI), income, and raised blood pressure. However, elevated stress was associated with higher infant DNA methylation at the MEST DMR (2.8% difference, P < 0.01) after adjusting for PTB. Maternal stress may be associated with epigenetic changes at MEST, a gene relevant to maternal care and obesity. Reduced prenatal stress may support the epigenomic profile of a healthy infant.

Erythrocyte folate concentrations, CpG methylation at genomically imprinted domains, and birth weight in a multiethnic newborn cohort.

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (ß = 143.2, se = 63.2, P = 0.02), third (ß = 117.3, se = 64.0, P = 0.07), and fourth (ß = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.

A paternal environmental legacy: evidence for epigenetic inheritance through the male germ line.

Literature on maternal exposures and the risk of epigenetic changes or diseases in the offspring is growing. Paternal contributions are often not considered. However, some animal and epidemiologic studies on various contaminants, nutrition, and lifestyle-related conditions suggest a paternal influence on the offspring's future health. The phenotypic outcomes may have been attributed to DNA damage or mutations, but increasing evidence shows that the inheritance of environmentally induced functional changes of the genome, and related disorders, are (also) driven by epigenetic components. In this essay we suggest the existence of epigenetic windows of susceptibility to environmental insults during sperm development. Changes in DNA methylation, histone modification, and non-coding RNAs are viable mechanistic candidates for a non-genetic transfer of paternal environmental information, from maturing germ cell to zygote. Inclusion of paternal factors in future research will ultimately improve the understanding of transgenerational epigenetic plasticity and health-related effects in future generations.

Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.