The Association of Childhood Maltreatment With Lipid Peroxidation and DNA Damage in Postpartum Women

Childhood maltreatment (CM) is associated with an increased risk for the development of psychiatric and somatic disorders in later life. A potential link could be oxidative stress, which is defined as the imbalance between the amount of reactive oxygen species (ROS) and the neutralizing capacity of anti-oxidative defense systems. However, the findings linking CM with oxidative stress have been inconsistent so far. In this study, we aimed to further explore this association by investigating biological markers of DNA and lipid damage due to oxidation in a comprehensive approach over two study cohorts of postpartum women (study cohort I and study cohort II). The severity of CM experiences (maltreatment load) was assessed in both studies using the Childhood Trauma Questionnaire. In study cohort I (N = 30), we investigated whether CM was associated with higher levels of structural DNA damage in peripheral blood mononuclear cells (PBMC) by two methods that are highly sensitive for detecting nuclear DNA strand breaks (comet assay and γH2AX staining). In study cohort II (N = 117), we then assessed in a larger cohort, that was specifically controlled for potential confounders for oxidative stress measurements, two established serum and plasma biomarkers of oxidative stress, one representing oxidative DNA and RNA damage (8-hydroxy-2′-deoxyguanosine and 8-hydroxyguanosine; 8-OH(d)G) and the other representing lipid peroxidation (8-isoprostane). In study cohort I, the analyses revealed no significant main effects of maltreatment load on cellular measures of nuclear DNA damage. The analyses of peripheral oxidative stress biomarkers in study cohort II revealed a significant main effect of maltreatment load on free 8-isoprostane plasma levels, but not on total 8-isprostane plasma levels and 8-OH(d)G serum levels. Taken together, by combining different methods and two study cohorts, we found no indications for higher oxidative DNA damages with higher maltreatment load in postpartum women. Further research is needed to investigate whether this increase in free 8-isoprostane is a marker for oxidative stress or whether it is instead functionally involved in ROS-related signaling pathways that potentially regulate inflammatory processes following a history of CM

The DNA methylation landscape of the human oxytocin receptor gene (OXTR): data-driven clusters and their relation to gene expression and childhood adversity.

The oxytocin receptor gene (OXTR) is of interest when investigating the effects of early adversity on DNA methylation. However, there is heterogeneity regarding the selection of the most promising CpG sites to target for analyses. The goal of this study was to determine functionally relevant clusters of CpG sites within the OXTR CpG island in 113 mother-infant dyads, with 58 of the mothers reporting childhood maltreatment (CM). OXTR DNA methylation was analyzed in peripheral/umbilical blood mononuclear cells. Different complexity reduction approaches were used to reduce the 188 CpG sites into clusters of co-methylated sites. Furthermore, associations between OXTR DNA methylation (cluster- and site-specific level) and OXTR gene expression and CM were investigated in mothers. Results showed that, first, CpG sections differed strongly regarding their statistical utility for research of individual differences in DNA methylation. Second, cluster analyses and Partial Least Squares (PLS) suggested two clusters consisting of intron1/exon2 and the protein-coding region of exon3, respectively, as most strongly associated with outcome measures. Third, cross-validated PLS regression explained 7% of variance in CM, with low cross-validated variance explained for the prediction of gene expression. Fourth, substantial mother-child correspondence was observed in correlation patterns within the identified clusters, but only modest correspondence outside these clusters. This study makes an important contribution to the mapping of the DNA methylation landscape of the OXTR CpG island by highlighting clusters of CpG sites that show desirable statistical properties and predictive value. We provide a Companion Web Application to facilitate the choice of CpG sites

Associations between childhood maltreatment and DNA methylation of the oxytocin receptor gene in immune cells of mother–newborn dyads

The neuropeptide oxytocin (OXT) and its receptor (OXTR) modulate interpersonal relationships, particularly mother-child interactions. DNA methylation (DNAm) changes of the OXTR gene were observed in individuals who experienced Childhood Maltreatment (CM). A modulatory role of single nucleotide polymorphisms (SNP) within OXTR in association with CM on the regulation of OXTR was also postulated. Whether these CM-induced epigenetic alterations are biologically inherited by the offspring remains unknown. We thus investigated possible intergenerational effects of maternal CM exposure on DNAm and OXTR gene expression, additionally accounting for the possible influence of three SNP: rs53576 and rs2254298 (OXTR gene), and rs2740210 (OXT gene). We used the Childhood Trauma Questionnaire to classify mothers into individuals with (CM+) or without CM (CM-). Maternal peripheral immune cells were isolated from venous blood (N = 117) and fetal immune cells from the umbilical cord (N = 113) after parturition. DNA methylation was assessed using MassARRAY. Taqman assays were performed for genotyping and gene expression analyses. Among mothers, CM was not associated with OXTR mean methylation or gene expression. However, four CpG sites showed different methylation levels in CM- compared to CM+. In mothers, the OXTR rs53576 and OXT rs2740210 allelic variations interacted with CM load on the OXTR mean methylation. Maternal and newborns' mean methylation of OXTR were positively associated within CM- dyads, but not in CM+ dyads. We show genexenvironment interactions on the epigenetic regulation of the oxytocinergic signaling and show the intergenerational comparability of the OXTR DNAm might be altered in infants of CM+ mothers

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation.Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D).Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women.Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system

The DNA methylation landscape of the human oxytocin receptor gene (OXTR): data-driven clusters and their relation to gene expression and childhood adversity

Abstract The oxytocin receptor gene (OXTR) is of interest when investigating the effects of early adversity on DNA methylation. However, there is heterogeneity regarding the selection of the most promising CpG sites to target for analyses. The goal of this study was to determine functionally relevant clusters of CpG sites within the OXTR CpG island in 113 mother-infant dyads, with 58 of the mothers reporting childhood maltreatment (CM). OXTR DNA methylation was analyzed in peripheral/umbilical blood mononuclear cells. Different complexity reduction approaches were used to reduce the 188 CpG sites into clusters of co-methylated sites. Furthermore, associations between OXTR DNA methylation (cluster- and site-specific level) and OXTR gene expression and CM were investigated in mothers. Results showed that, first, CpG sections differed strongly regarding their statistical utility for research of individual differences in DNA methylation. Second, cluster analyses and Partial Least Squares (PLS) suggested two clusters consisting of intron1/exon2 and the protein-coding region of exon3, respectively, as most strongly associated with outcome measures. Third, cross-validated PLS regression explained 7% of variance in CM, with low cross-validated variance explained for the prediction of gene expression. Fourth, substantial mother-child correspondence was observed in correlation patterns within the identified clusters, but only modest correspondence outside these clusters. This study makes an important contribution to the mapping of the DNA methylation landscape of the OXTR CpG island by highlighting clusters of CpG sites that show desirable statistical properties and predictive value. We provide a Companion Web Application to facilitate the choice of CpG sites

High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington’s disease expansion mutation carriers shows normal mitochondrial respiratory function

<div><p>Alterations in mitochondrial respiration are an important hallmark of Huntington’s disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (Hdh^Q111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the Hdh^Q111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.</p></div

Clinical data of the human study cohort.

<p>Clinical data of the human study cohort.</p

Mouse primers for qPCR.

<p>Mouse primers for qPCR.</p

Respiration of cortex, liver, soleus muscle and heart of Hdh^Q20 and Hdh^Q111 mice.

<p>High-resolution respirometry in the murine tissues prefrontal cortex, liver, soleus muscle and heart. The white boxes represent Hdh^Q20 control mice versus the gray boxes, which represent the HD mouse model Hdh^Q111. (A) Complex I activity (C_I) is shown in a coupled state determined after addition of ADP. (B) Complex II activity (C_II) was measured in an uncoupled state after addition of rotenone. (C) The <i>maximum OxPhos</i> capacity was measured and (D) the <i>maximum uncoupled capacity</i> was determined after application of FCCP in all four tissues. (E) For murine cortex and liver the oxygen consumption linked to ATP production (O₂ATP) was calculated. n = 6, student’s t-test comparing genotypes, ns = p>0.05, * = p≤0.05, ** = p≤0.01. 95% Confidence interval limits: (B) liver: 14.72–65.71; (D) cortex: 1.20–28.16; liver: 4.99–73.43.; (E) cortex: 0.96–13.67. The Mann-Whitney Rank-Test was used for the following samples: (A): cortex; (B): cortex, m. soleus, heart; (D): m. soleus.</p

Representative high-resolution respirometry recordings of the human vastus lateralis muscle and the murine prefrontal cortex and heart.

<p>Representative measurements of high-resolution respirometry recordings are shown for the human vastus lateralis muscle (A), the murine prefrontal cortex (B) and the murine heart (C). The blue line represents the oxygen concentration in the chamber, while the red line indicates the oxygen flux. (A) For experiments with human vastus lateralis muscle samples 10 mM pyruvate were added before the recording started. After administration of 5 mM malate (M), 10 mM glutamate (G) and 5 mM ADP complex I activity (CI) was recorded and the leak respiration was calculated (respiration after MG/respiration after ADP). 10 μM cytochrome c was added to survey the integrity of the outer mitochondrial membrane. 1 mM octanoyl carnitine was administered to measure the fatty acid induced respiration. Further addition of 10 mM succinate, which is the complex II substrate, enables the determination of the maximum OxPhos capacity. The ATP synthase inhibitor oligomycin (O) (5 μM), which was not used for the murine heart (C) and soleus muscle was administered. After adding the uncoupling agent carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (0.5, 0.25, 0.25 μM FCCP) the maximum uncoupled capacity was measured. Finally complex I was inhibited by rotenone (R) (0.5 μM) allowing the determination of complex II activity (CII). In the end complex III was inhibited by administration of antimycin A (5 μM). (B) The protocol for murine cortex and liver matched the human muscle protocol with the following exceptions: cytochrome c was added after pyruvate before the recording started and octanoyl carnitine was not used. (C) The protocol for murine heart and soleus muscle is equivalent to the one used for murine cortex (B) except for the oligomycin administration.</p