Antidepressant Exposure and DNA Methylation: Insights from a Methylome-Wide Association Study.

Importance: Understanding antidepressant mechanisms could help design more effective and tolerated treatments. Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. Design: Case-control methylome-wide association studies (MWAS) of antidepressant exposure were performed from blood samples collected between 2006-2011 in Generation Scotland (GS). The summary statistics were tested for enrichment in specific tissues, gene ontologies and an independent MWAS in the Netherlands Study of Depression and Anxiety (NESDA). A methylation profile score (MPS) was derived and tested for its association with antidepressant exposure in eight independent cohorts, alongside prospective data from GS. Setting: Cohorts; GS, NESDA, FTC, SHIP-Trend, FOR2107, LBC1936, MARS-UniDep, ALSPAC, E-Risk, and NTR. Participants: Participants with DNAm data and self-report/prescription derived antidepressant exposure. Main Outcomes and Measures: Whole-blood DNAm levels were assayed by the EPIC/450K Illumina array (9 studies, N exposed = 661, N unexposed = 9,575) alongside MBD-Seq in NESDA (N exposed = 398, N unexposed = 414). Antidepressant exposure was measured by self- report and/or antidepressant prescriptions. Results: The self-report MWAS (N = 16,536, N exposed = 1,508, mean age = 48, 59% female) and the prescription-derived MWAS (N = 7,951, N exposed = 861, mean age = 47, 59% female), found hypermethylation at seven and four DNAm sites (p < 9.42x10 -8 ), respectively. The top locus was cg26277237 ( KANK1, p self-report = 9.3x10 -13 , p prescription = 6.1x10 -3 ). The self-report MWAS found a differentially methylated region, mapping to DGUOK-AS1 ( p adj = 5.0x10 -3 ) alongside significant enrichment for genes expressed in the amygdala, the "synaptic vesicle membrane" gene ontology and the top 1% of CpGs from the NESDA MWAS (OR = 1.39, p < 0.042). The MPS was associated with antidepressant exposure in meta-analysed data from external cohorts (N studies = 9, N = 10,236, N exposed = 661, f3 = 0.196, p < 1x10 -4 ). Conclusions and Relevance: Antidepressant exposure is associated with changes in DNAm across different cohorts. Further investigation into these changes could inform on new targets for antidepressant treatments. 3 Key Points: Question: Is antidepressant exposure associated with differential whole blood DNA methylation?Findings: In this methylome-wide association study of 16,536 adults across Scotland, antidepressant exposure was significantly associated with hypermethylation at CpGs mapping to KANK1 and DGUOK-AS1. A methylation profile score trained on this sample was significantly associated with antidepressant exposure (pooled f3 [95%CI]=0.196 [0.105, 0.288], p < 1x10 -4 ) in a meta-analysis of external datasets. Meaning: Antidepressant exposure is associated with hypermethylation at KANK1 and DGUOK-AS1 , which have roles in mitochondrial metabolism and neurite outgrowth. If replicated in future studies, targeting these genes could inform the design of more effective and better tolerated treatments for depression.

Influence of glycopeptide antibiotics on purine metabolism of endothelial cells.

We provide evidence that the commercially available preparations of glycopeptides for intravenous application are well tolerated by endothelial cells when applied in concentrations less than 5 mg/ml. Since the antibiotics tested are administered at maximal concentrations of 10 mg/ml, the dose range used in our in vitro experiments (5 and 10 mg/ml) mimics possible clinical concentrations at the site of infusion. Similar concentrations may be reached by retrograde intravenous pressure infusion techniques (10-12). We have demonstrated that these high concentrations lead to considerable endothelial cell damage. These findings may explain the common side effect associated with intravenously applied glycopeptides namely pain and phlebitis at the site of infusion (2, 13). Figure 1 shows that a detrimental effect measurable after 20 min occurs only using vancomycin solutions at concentrations of 10 mg/ml, whereas already a dilution to 5 mg/ml renders the solutions more compatible to HUVEC. These data are in line with the observation that slow intravenous application of glycopeptides into large veins can largely prevent the occurrence of local phlebitis. Alternatively, the occurrence of phlebitis should be avoidable by diluting the manufacturers' preparations at least to 2-5 mg/ml and not 10 mg/ml as recommended by the manufacturer of vancomycin. The same aspects need to be considered for use of glycopeptides for retrograde high pressure infusion. The tolerance of intravenously applied antibiotics has previously been tested in animal models (4). Our model of human venous endothelial cells for testing antibiotic solutions for intravenous compatibility provides a valuable alternate model. In conclusion our data show that the commercial preparation of teicoplanin is more compatible for HUVEC than those of vancomycin.

Endothelial cell compatibility of glycopeptide antibiotics for intravenous use.

The use of human venous endothelial cells for testing antibiotic solutions for intravenous compatibility provides a valuable alternative to animal models. In order to evaluate the effect of vancomycin and teicoplanin on the viability of human umbilical venous endothelial cells, intracellular ATP levels were measured by a luciferin-luciferase assay. Prostacyclin (PGI2) and thromboxane A2 (TXA2) were determined by direct radioimmunoassay. Vancomycin at concentrations of 5 and 10 mg/mL reduced the intracellular ATP content by 18.7% and 69.9%, respectively, within 60 min. In contrast, cellular energy charge remained significantly higher after incubation with teicoplanin at 5 and 10 mg/mL (reduction 8.7% and 15.5%, respectively). Neither vancomycin nor teicoplanin at a concentration of 2 mg/mL led to significant ATP decline. However, endothelial cells incubated with vancomycin resulted in significantly lower release of PGI2 and TXA2 compared with teicoplanin. These results show that teicoplanin is more compatible with endothelial cells than vancomycin, and that both antibiotics are well tolerated if diluted to a final concentration of 2 mg/mL.

Effect of single oral dose of azithromycin, clarithromycin, and roxithromycin on polymorphonuclear leukocyte function assessed ex vivo by flow cytometry.

Azithromycin was given as a single oral dose (20 mg/kg of body weight) to 12 volunteers in a crossover study with roxithromycin (8 to 12 mg/kg) and clarithromycin (8 to 12 mg/kg). Flow cytometry was used to study the phagocytic functions and the release of reactive oxygen products following phagocytosis by neutrophil granulocytes prior to administration of the three drugs, 16 h after azithromycin administration, and 3 h after clarithromycin and roxithromycin administration. Phagocytic capacity was assessed by measuring the uptake of fluorescein isothiocyanate-labeled bacteria. Reactive oxygen generation after phagocytosis of unlabeled bacteria was estimated by the amount of dihydrorhodamine 123 converted to rhodamine 123 intracellularly. Azithromycin resulted in decreased capacities of the cells to phagocytize Escherichia coli (median [range], 62% [27 to 91%] of the control values; P < 0.01) and generate reactive oxygen products (75% [34 to 26%] of the control values; P < 0.01). Clarithromycin resulted in reduced phagocytosis (82% [75 to 98%] of control values; P < 0.01) but did not alter reactive oxygen production (84% [63 to 113%] of the control values; P > 0.05). Roxithromycin treatment did not affect granulocyte phagocytosis (92% [62 to 118%] of the control values; P > 0.05) or reactive oxygen production (94% [66 to 128%] of the control value; P > 0.05). No relation between intra- and/or extracellular concentrations of azithromycin and/or roxithromycin and the polymorphonuclear phagocyte function and/or reactive oxygen production existed (P > 0.05 for all comparisons). These results demonstrate that the accumulation of macrolides in neutrophils can suppress the response of phagocytic cells to bacterial pathogens after a therapeutic dose.