Ketamine Produces Antidepressant Effects by Inhibiting Histone Deacetylases and Upregulating Hippocampal Brain-Derived Neurotrophic Factor Levels in a Diisopropyl Fluorophosphate-Based Rat Model of Gulf War Illness.

Approximately one-third of Gulf War veterans suffer from Gulf War Illness (GWI), which encompasses mood disorders and depressive symptoms. Deployment-related exposure to organophosphate compounds has been associated with GWI development. Epigenetic modifications have been reported in GWI veterans. We previously showed that epigenetic histone dysregulations were associated with decreased brain-derived neurotrophic factor (BDNF) expression in a GWI rat model. GWI has no effective therapies. Ketamine (KET) has recently been approved by the Food and Drug Administration for therapy-resistant depression. Interestingly, BDNF upregulation underlies KET's antidepressant effect in GWI-related depression. Here, we investigated whether KET's effect on histone mechanisms signals BDNF upregulations in GWI. Male Sprague-Dawley rats were injected once daily with diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c., 5 days). At 6 months following DFP exposure, KET (10 mg/kg, i.p.) was injected, and brains were dissected 24 hours later. Western blotting was used for protein expression, and epigenetic studies used chromatin immunoprecipitation methods. Dil staining was conducted for assessing dendritic spines. Our results indicated that an antidepressant dose of KET inhibited the upregulation of histone deacetylase (HDAC) enzymes in DFP rats. Furthermore, KET restored acetylated histone occupancy at the Bdnf promoter IV and induced BDNF protein expression in DFP rats. Finally, KET treatment also increased the spine density and altered the spine diversity with increased T-type and decreased S-type spines in DFP rats. Given these findings, we propose that KET's actions involve the inhibition of HDAC expression, upregulation of BDNF, and dendritic modifications that together ameliorates the pathologic synaptic plasticity and exerts an antidepressant effect in DFP rats. SIGNIFICANCE STATEMENT: This study offers evidence supporting the involvement of epigenetic histone pathways in the antidepressant effects of ketamine (KET) in a rat model of Gulf War Illness (GWI)-like depression. This effect is achieved through the modulation of histone acetylation at the Bdnf promoter, resulting in elevated brain-derived neurotrophic factor expression and subsequent dendritic remodeling in the hippocampus. These findings underscore the rationale for considering KET as a potential candidate for clinical trials aimed at managing GWI-related depression.

Epigenetics in drug disposition & drug therapy: symposium report of the 24th North American meeting of the International Society for the Study of Xenobiotics (ISSX).

The 24th North American International Society for the Study of Xenobiotics (ISSX) meeting, held virtually from September 13 to 17, 2021, embraced the theme of "Broadening Our Horizons." This reinforces a key mission of ISSX: striving to share innovative science related to drug discovery and development. Session speakers and the ISSX New Investigators Group, which supports the scientific and professional development of student and early career ISSX members, elected to highlight the scientific content presented during the captivating session titled, "Epigenetics in Drug Disposition & Drug Therapy." The impact genetic variation has on drug response is well established; however, this session underscored the importance of investigating the role of epigenetics in drug disposition and drug discovery. Session speakers, Drs. Ning, McClay, and Lazarus, detailed mechanisms by which epigenetic players including long non-coding RNA (lncRNAs), microRNA (miRNAs), DNA methylation, and histone acetylation can alter the expression of genes involved in pharmacokinetics, pharmacodynamics, and toxicity. Dr. Ning detailed current knowledge about miRNAs and lncRNAs and the mechanisms by which they can affect the expression of drug metabolizing enzymes (DMEs) and nuclear receptors. Dr. Lazarus discussed the potential role of miRNAs on UDP-glucuronosyltransferase (UGT) expression and activity. Dr. McClay provided evidence that aging alters methylation and acetylation of DMEs in the liver, affecting gene expression and activity. These topics, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are herein discussed, along with exciting future perspectives for epigenetics in drug disposition and drug discovery research.

Histone acetylation at the sulfotransferase 1a1 gene is associated with its hepatic expression in normal aging.

OBJECTIVES: Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects. METHODS: We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months. RESULTS: Our sample shows a significant loss of 5mC at Sult1a1 (ß = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (ß = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6. CONCLUSIONS: Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults.

Review and Consensus on Pharmacogenomic Testing in Psychiatry.

The implementation of pharmacogenomic (PGx) testing in psychiatry remains modest, in part due to divergent perceptions of the quality and completeness of the evidence base and diverse perspectives on the clinical utility of PGx testing among psychiatrists and other healthcare providers. Recognizing the current lack of consensus within the field, the International Society of Psychiatric Genetics assembled a group of experts to conduct a narrative synthesis of the PGx literature, prescribing guidelines, and product labels related to psychotropic medications as well as the key considerations and limitations related to the use of PGx testing in psychiatry. The group concluded that to inform medication selection and dosing of several commonly-used antidepressant and antipsychotic medications, current published evidence, prescribing guidelines, and product labels support the use of PGx testing for 2 cytochrome P450 genes (CYP2D6, CYP2C19). In addition, the evidence supports testing for human leukocyte antigen genes when using the mood stabilizers carbamazepine (HLA-A and HLA-B), oxcarbazepine (HLA-B), and phenytoin (CYP2C9, HLA-B). For valproate, screening for variants in certain genes (POLG, OTC, CSP1) is recommended when a mitochondrial disorder or a urea cycle disorder is suspected. Although barriers to implementing PGx testing remain to be fully resolved, the current trajectory of discovery and innovation in the field suggests these barriers will be overcome and testing will become an important tool in psychiatry.

Building a schizophrenia genetic network: transcription factor 4 regulates genes involved in neuronal development and schizophrenia risk.

The transcription factor 4 (TCF4) locus is a robust association finding with schizophrenia (SCZ), but little is known about the genes regulated by the encoded transcription factor. Therefore, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 in neural-derived (SH-SY5Y) cells to identify genome-wide TCF4 binding sites, followed by data integration with SCZ association findings. We identified 11 322 TCF4 binding sites overlapping in two ChIP-seq experiments. These sites are significantly enriched for the TCF4 Ebox binding motif (>85% having ≥1 Ebox) and implicate a gene set enriched for genes downregulated in TCF4 small-interfering RNA (siRNA) knockdown experiments, indicating the validity of our findings. The TCF4 gene set was also enriched among (1) gene ontology categories such as axon/neuronal development, (2) genes preferentially expressed in brain, in particular pyramidal neurons of the somatosensory cortex and (3) genes downregulated in postmortem brain tissue from SCZ patients (odds ratio, OR = 2.8, permutation P < 4x10-5). Considering genomic alignments, TCF4 binding sites significantly overlapped those for neural DNA-binding proteins such as FOXP2 and the SCZ-associated EP300. TCF4 binding sites were modestly enriched among SCZ risk loci from the Psychiatric Genomic Consortium (OR = 1.56, P = 0.03). In total, 130 TCF4 binding sites occurred in 39 of the 108 regions published in 2014. Thirteen genes within the 108 loci had both a TCF4 binding site ±10kb and were differentially expressed in siRNA knockdown experiments of TCF4, suggesting direct TCF4 regulation. These findings confirm TCF4 as an important regulator of neural genes and point toward functional interactions with potential relevance for SCZ.

Effects of HIV-1 Tat and Methamphetamine on Blood-Brain Barrier Integrity and Function In Vitro.

Human immunodeficiency (HIV) infection results in neurocognitive deficits in about one half of infected individuals. Despite systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting central nervous system (CNS)-localized infection. Drug abuse exacerbates HIV-induced cognitive and pathological CNS changes. This study's purpose was to investigate the effects of the HIV-1 protein Tat and methamphetamine on factors affecting drug penetration across an in vitro BBB model. Factors affecting paracellular and transcellular flux in the presence of Tat and methamphetamine were examined. Transendothelial electrical resistance, ZO-1 expression, and lucifer yellow (a paracellular tracer) flux were aspects of paracellular processes that were examined. Additionally, effects on P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP-1) mRNA (via quantitative PCR [qPCR]) and protein (via immunoblotting) expression were measured; Pgp and MRP-1 are drug efflux proteins. Transporter function was examined after exposure of Tat with or without methamphetamine using the P-gp substrate rhodamine 123 and also using the dual P-gp/MRP-1 substrate and protease inhibitor atazanavir. Tat and methamphetamine elicit complex changes affecting transcellular and paracellular transport processes. Neither Tat nor methamphetamine significantly altered P-gp expression. However, Tat plus methamphetamine exposure significantly increased rhodamine 123 accumulation within brain endothelial cells, suggesting that treatment inhibited or impaired P-gp function. Intracellular accumulation of atazanavir was not significantly altered after Tat or methamphetamine exposure. Atazanavir accumulation was, however, significantly increased by simultaneous inhibition of P-gp and MRP. Collectively, our investigations indicate that Tat and methamphetamine alter aspects of BBB integrity without affecting net flux of paracellular compounds. Tat and methamphetamine may also affect several aspects of transcellular transport.

Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

BACKGROUND: Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. METHODS: We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. RESULTS: No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10-5 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10-5 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. CONCLUSIONS: To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD.

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects.

The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25-92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10(-8)). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10(-30)). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.

Deep Sequencing of Three Loci Implicated in Large-Scale Genome-Wide Association Study Smoking Meta-Analyses.

INTRODUCTION: Genome-wide association study meta-analyses have robustly implicated three loci that affect susceptibility for smoking: CHRNA5\CHRNA3\CHRNB4, CHRNB3\CHRNA6 and EGLN2\CYP2A6. Functional follow-up studies of these loci are needed to provide insight into biological mechanisms. However, these efforts have been hampered by a lack of knowledge about the specific causal variant(s) involved. In this study, we prioritized variants in terms of the likelihood they account for the reported associations. METHODS: We employed targeted capture of the CHRNA5\CHRNA3\CHRNB4, CHRNB3\CHRNA6, and EGLN2\CYP2A6 loci and flanking regions followed by next-generation deep sequencing (mean coverage 78×) to capture genomic variation in 363 individuals. We performed single locus tests to determine if any single variant accounts for the association, and examined if sets of (rare) variants that overlapped with biologically meaningful annotations account for the associations. RESULTS: In total, we investigated 963 variants, of which 71.1% were rare (minor allele frequency < 0.01), 6.02% were insertion/deletions, and 51.7% were catalogued in dbSNP141. The single variant results showed that no variant fully accounts for the association in any region. In the variant set results, CHRNB4 accounts for most of the signal with significant sets consisting of directly damaging variants. CHRNA6 explains most of the signal in the CHRNB3\CHRNA6 locus with significant sets indicating a regulatory role for CHRNA6. Significant sets in CYP2A6 involved directly damaging variants while the significant variant sets suggested a regulatory role for EGLN2. CONCLUSIONS: We found that multiple variants implicating multiple processes explain the signal. Some variants can be prioritized for functional follow-up.

Refinement of schizophrenia GWAS loci using methylome-wide association data.

Recent genome-wide association studies (GWAS) have made substantial progress in identifying disease loci. The next logical step is to design functional experiments to identify disease mechanisms. This step, however, is often hampered by the large size of loci identified in GWAS that is caused by linkage disequilibrium between SNPs. In this study, we demonstrate how integrating methylome-wide association study (MWAS) results with GWAS findings can narrow down the location for a subset of the putative casual sites. We use the disease schizophrenia as an example. To handle "data analytic" variation, we first combined our MWAS results with two GWAS meta-analyses (N = 32,143 and 21,953), that had largely overlapping samples but different data analysis pipelines, separately. Permutation tests showed significant overlapping association signals between GWAS and MWAS findings. This significant overlap justified prioritizing loci based on the concordance principle. To further ensure that the methylation signal was not driven by chance, we successfully replicated the top three methylation findings near genes SDCCAG8, CREB1 and ATXN7 in an independent sample using targeted pyrosequencing. In contrast to the SNPs in the selected region, the methylation sites were largely uncorrelated explaining why the methylation signals implicated much smaller regions (median size 78 bp). The refined loci showed considerable enrichment of genomic elements of possible functional importance and suggested specific hypotheses about schizophrenia etiology. Several hypotheses involved possible variation in transcription factor-binding efficiencies.

Combined Whole Methylome and Genomewide Association Study Implicates CNTN4 in Alcohol Use.

BACKGROUND: Methylome-wide association (MWAS) studies present a new way to advance the search for biological correlates for alcohol use. A challenge with methylation studies of alcohol involves the causal direction of significant methylation-alcohol associations. One way to address this issue is to combine MWAS data with genomewide association study (GWAS) data. METHODS: Here, we combined MWAS and GWAS results for alcohol use from 619 individuals. Our MWAS data were generated by next-generation sequencing of the methylated genomic DNA fraction, producing over 60 million reads per subject to interrogate methylation levels at ~27 million autosomal CpG sites in the human genome. Our GWAS included 5,571,786 single nucleotide polymorphisms (SNPs) imputed with 1000 Genomes. RESULTS: When combining the MWAS and GWAS data, our top finding was a region in an intron of CNTN4 (p = 2.55 × 10(-8) ), located between chr3: 2,555,403 and 2,555,524, encompassing SNPs rs1382874 and rs1382875. This finding was then replicated in an independent sample of 730 individuals. We used bisulfite pyrosequencing to measure methylation and found significant association with regular alcohol use in the same direction as the MWAS (p = 0.021). Rs1382874 and rs1382875 were genotyped and found to be associated in the same direction as the GWAS (p = 0.008 and p = 0.009). After integrating the MWAS and GWAS findings from the replication sample, we replicated our combined analysis finding (p = 0.0017) in CNTN4. CONCLUSIONS: Through combining methylation and SNP data, we have identified CNTN4 as a risk factor for regular alcohol use.

Epigenetic regulation of drug metabolism in aging: utilizing epigenetics to optimize geriatric pharmacotherapy.

We explore the relationship between epigenetic aging and drug metabolism. We review current evidence for changes in drug metabolism in normal aging, followed by a description of how epigenetic modifications associated with age can regulate the expression and functionality of genes. In particular, we focus on the role of epigenome-wide studies of human and mouse liver in understanding these age-related processes with respect to xenobiotic processing. We highlight genes encoding drug metabolizing enzymes and transporters revealed to be affected by epigenetic aging in these studies. We conclude that substantial evidence exists for epigenetic aging impacting drug metabolism and transport genes, but more work is needed. We further highlight the promise of pharmacoepigenetics applied to enhancing drug safety in older adults.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Estimation of CpG coverage in whole methylome next-generation sequencing studies.

BACKGROUND: Methylation studies are a promising complement to genetic studies of DNA sequence. However, detailed prior biological knowledge is typically lacking, so methylome-wide association studies (MWAS) will be critical to detect disease relevant sites. A cost-effective approach involves the next-generation sequencing (NGS) of single-end libraries created from samples that are enriched for methylated DNA fragments. A limitation of single-end libraries is that the fragment size distribution is not observed. This hampers several aspects of the data analysis such as the calculation of enrichment measures that are based on the number of fragments covering the CpGs. RESULTS: We developed a non-parametric method that uses isolated CpGs to estimate sample-specific fragment size distributions from the empirical sequencing data. Through simulations we show that our method is highly accurate. While the traditional (extended) read count methods resulted in severely biased coverage estimates and introduces artificial inter-individual differences, through the use of the estimated fragment size distributions we could remove these biases almost entirely. Furthermore, we found correlations of 0.999 between coverage estimates obtained using fragment size distributions that were estimated with our method versus those that were "observed" in paired-end sequencing data. CONCLUSIONS: We propose a non-parametric method for estimating fragment size distributions that is highly precise and can improve the analysis of cost-effective MWAS studies that sequence single-end libraries created from samples that are enriched for methylated DNA fragments.

Genome-wide association study of patient-rated and clinician-rated global impression of severity during antipsychotic treatment.

OBJECTIVE: To examine the unique and congruent findings between multiple raters in a genome-wide association study (GWAS) in the context of understanding individual differences in treatment response during antipsychotic therapy for schizophrenia. MATERIALS AND METHODS: We performed GWAS to search for genetic variation affecting treatment response. The analysis sample included 738 patients with schizophrenia, successfully genotyped for ∼492k single nucleotide polymorphisms (SNPs) from the Clinical Antipsychotic Trial of Intervention Effectiveness. Outcomes included both clinician and patient report of illness severity on global impression scales, the clinical global impression severity scale and patient global impression, respectively. Our criterion for genome-wide significance was a prespecified threshold ensuring that, on average, only 10% of the significant findings are false discoveries. RESULTS: Thirteen SNPs reached genome-wide significance. The top findings indicated three SNPs in PDE4D, 5q12.1 (P=4.2×10, 1.6×10, 1.8×10), mediating the effects of quetiapine on patient-reported severity and an additional three SNPs in TJP1, 15q13.1 (P=2.25×10, 4.86×10, 4.91×10), mediating the effects of risperidone on patient-reported severity. For clinician-reported severity, two SNPs in PPA2, 4q24 (P=3.68×10, 5.05×10), were found to reach genome-wide significance. CONCLUSION: We found evidence of both a novel and a consistent association when examining the results from the patient and clinician ratings, suggesting that different raters may capture unique facets of schizophrenia. Although our findings require replication and functional validation, this study shows the potential of GWAS to discover genes that potentially mediate treatment response of antipsychotic medication.

Genes, environments, and developmental research: methods for a multi-site study of early substance abuse.

The importance of including developmental and environmental measures in genetic studies of human pathology is widely acknowledged, but few empirical studies have been published. Barriers include the need for longitudinal studies that cover relevant developmental stages and for samples large enough to deal with the challenge of testing gene-environment-development interaction. A solution to some of these problems is to bring together existing data sets that have the necessary characteristics. As part of the National Institute on Drug Abuse-funded Gene-Environment-Development Initiative, our goal is to identify exactly which genes, which environments, and which developmental transitions together predict the development of drug use and misuse. Four data sets were used of which common characteristics include (1) general population samples, including males and females; (2) repeated measures across adolescence and young adulthood; (3) assessment of nicotine, alcohol, and cannabis use and addiction; (4) measures of family and environmental risk; and (5) consent for genotyping DNA from blood or saliva. After quality controls, 2,962 individuals provided over 15,000 total observations. In the first gene-environment analyses, of alcohol misuse and stressful life events, some significant gene-environment and gene-development effects were identified. We conclude that in some circumstances, already collected data sets can be combined for gene-environment and gene-development analyses. This greatly reduces the cost and time needed for this type of research. However, care must be taken to ensure careful matching across studies and variables.

The influence of five monoamine genes on trajectories of depressive symptoms across adolescence and young adulthood.

The influence of five monoamine candidate genes on depressive symptom trajectories in adolescence and young adulthood were examined in the Add Health genetic sample. Results indicated that, for all respondents, carriers of the dopamine receptor D4 5-repeat allele were characterized by distinct depressive symptom trajectories across adolescence and early adulthood. Similarly, for males, individuals with the monoamine oxidase A 3.5-repeat allele exhibited unique depressive symptom trajectories. Specifically, the trajectories of those with the dopamine receptor D4 5-repeat allele were characterized by rising levels in the transition to adulthood, while their peers were experiencing a normative drop in depressive symptom frequency. Conversely, males with the monoamine oxidase A 3.5-repeat allele were shown to experience increased distress in late adolescence. An empirical method for examining a wide array of allelic combinations was employed, and false discovery rate methods were used to control the risk of false positives due to multiple testing. Special attention was given to thoroughly interrogate the robustness of the putative genetic effects. These results demonstrate the value of combining dynamic developmental perspectives with statistical genetic methods to optimize the search for genetic influences on psychopathology across the life course.

Repeated exposure to chlorpyrifos is associated with a dose-dependent chronic neurobehavioral deficit in adult rats.

Organophosphate (OP) chemicals include commonly used pesticides and chemical warfare agents, and mechanistically they are potent inhibitors of the cholinesterase (ChE) enzyme. Epidemiological studies report long-term neuropsychiatric issues, including depression and cognitive impairments in OP-exposed individuals. Chlorpyrifos (CPF) is one of the most widely used pesticides worldwide. Multiple laboratory studies have reported on either the long-term behavioral effect of an acute high-dose CPF (30-250 mg/kg) or studied sub-chronic behavioral effects, particularly the motor and cognitive effects of repeated low-dose CPF. However, studies are lacking on chronic mood and depression-related morbidities following repeated CPF doses that would mimic occupationally relevant OP exposures during adulthood. In this study, adult male rats were injected with CPF (1, 3, 5, or 10 mg/kg/d, s.c.) for 21 consecutive days. Dependent on the CPF dose, ChE activity was inhibited approximately 60-80% in the blood and about 20-50% in the hippocampus at 2-days after the end of CPF exposures. Following a 12-week washout period, a complete recovery of ChE activity was noted. However, CPF-treated rats exhibited a dose-dependent increase in signs related to anhedonia (sucrose preference test), anxiety (open-field and elevated plus-maze), and despair (forced swim test) at this stage. To the best of our knowledge, this could be the first laboratory study that demonstrates a cause-effect relationship between occupational-like CPF exposures in adult rats and the development of long-term depression-related outcomes and could provide an experimental system to study molecular mechanisms underlying environmental OP exposures and the elevated risk for chronic behavioral deficits.

Epigenetic histone acetylation and Bdnf dysregulation in the hippocampus of rats exposed to repeated, low-dose diisopropylfluorophosphate.

AIMS: Deployment-related exposures to organophosphate (OP) compounds are implicated for Gulf War Illness (GWI) development in First GW veterans. However, reasons for the persistence of GWI are not fully understood. Epigenetic modifications to chromatin are regulatory mechanisms that can adaptively or maladaptively respond to external stimuli. These include DNA methylation and histone acetylation. DNA methylation changes have been reported in GWI but the role of histone acetylation in GWI has been less explored, despite its importance as an epigenetic mechanism for neurological disorders. MAIN METHODS: Male Sprague-Dawley rats were exposed to OP diisopropyl fluorophosphate (DFP, 0.5 mg/kg s.c., 5-d) and 6-m later brains were dissected for hippocampus. Western blotting, activity assays and chromatin immunoprecipitation (ChIP) were utilized for epigenetic analyses. Behavior was assessed using the Forced Swim Test (FST) and the Elevated Plus Maze (EPM). KEY FINDINGS: We observed a significant upregulation in HDAC1 protein along with a significant increase in HDAC enzyme activity in the hippocampus of DFP rats. A locus-specific ChIP study revealed decreases in H3K9ac at the brain derived neurotrophic factor (Bdnf) promoter IV coupled with a significant decrease in BDNF protein in DFP rat hippocampus. Treatment with HDAC inhibitor valproic acid reduced HDAC activity and decreased the FST immobility time in DFP rats. SIGNIFICANCE: Our research suggests that epigenetic alterations to histone acetylation pathways and decreased BDNF expression could represent novel mechanisms for GWI symptomatology and may provide new targets for developing effective drugs for GWI treatment.

(1)H nuclear magnetic resonance metabolomics analysis identifies novel urinary biomarkers for lung function.

Chronic obstructive pulmonary disease (COPD), characterized by chronic airflow limitation, is a serious public health concern. In this study, we used proton nuclear magnetic resonance ((1)H NMR) spectroscopy to identify and quantify metabolites associated with lung function in COPD. Plasma and urine were collected from 197 adults with COPD and from 195 without COPD. Samples were assayed using a 600 MHz NMR spectrometer, and the resulting spectra were analyzed against quantitative spirometric measures of lung function. After correcting for false discoveries and adjusting for covariates (sex, age, smoking) several spectral regions in urine were found to be significantly associated with baseline lung function. These regions correspond to the metabolites trigonelline, hippurate and formate. Concentrations of each metabolite, standardized to urinary creatinine, were associated with baseline lung function (minimum p-value = 0.0002 for trigonelline). No significant associations were found with plasma metabolites. Urinary hippurate and formate are often related to gut microflora. This could suggest that the microbiome varies between individuals with different lung function. Alternatively, the associated metabolites may reflect lifestyle differences affecting overall health. Our results will require replication and validation, but demonstrate the utility of NMR metabolomics as a screening tool for identifying novel biomarkers of pulmonary outcomes.