In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

Oxidized in vitro genomic DNA (gDNA) is known to launch an adaptive response in human cell cultures. The cfDNA extracted from the plasma of schizophrenic patients (sz-cfDNA) and healthy controls (hc-cfDNA) contains increased amounts of 8-oxodG, a DNA-oxidation marker. The aim of the research was answering a question: can the human cfDNA isolated from blood plasma stimulate the adaptive response in human cells? In vitro responses of ten human skin fibroblasts (HSFs) and four peripheral blood mononuclear cell (PBMC) lines after 1-24 h of incubation with sz-cfDNA, gDNA and hc-cfDNA containing different amounts of 8-oxodG were examined. Expressions of RNA of eight genes (NOX4, NFE2L2, SOD1, HIF1A, BRCA1, BRCA2, BAX and BCL2), six proteins (NOX4, NRF2, SOD1, HIF1A, γH2AX and BRCA1) and DNA-oxidation marker 8-oxodG were analyzed by RT-qPCR and flow cytometry (when analyzing the data, a subpopulation of lymphocytes (PBL) was identified). Adding hc-cfDNA or sz-cfDNA to HSFs or PBMC media in equal amounts (50 ng/mL, 1-3 h) stimulated transient synthesis of free radicals (ROS), which correlated with an increase in the expressions of NOX4 and SOD1 genes and with an increase in the levels of the markers of DNA damage γH2AX and 8-oxodG. ROS and DNA damage induced an antioxidant response (expression of NFE2L2 and HIF1A), DNA damage response (BRCA1 and BRCA2 gene expression) and anti-apoptotic response (changes in BAX and BCL2 genes expression). Heterogeneity of cells of the same HSFs or PBL population was found with respect to the type of response to (sz,hc)-cfDNA. Most cells responded to oxidative stress with an increase in the amount of NRF2 and BRCA1 proteins along with a moderate increase in the amount of NOX4 protein and a low amount of 8-oxodG oxidation marker. However, upon the exposure to (sz,hc)-cfDNA, the size of the subpopulation with apoptosis signs (high DNA damage degree, high NOX4 and low NRF2 and BRCA1 levels) also increased. No significant difference between the responses to sz-cfDNA and hc-cfDNA was observed. Sz-cfDNA and hc-cfDNA showed similarly high bioactivity towards fibroblasts and lymphocytes. Conclusion: In cultured human cells, hc-cfDNA and sz-cfDNA equally stimulated an adaptive response aimed at launching the antioxidant, repair, and anti-apoptotic processes. The mediator of the development of the adaptive response are ROS produced by, among others, NOX4 and SOD1 enzymes.

Ribosomal DNA Abundance in the Patient's Genome as a Feasible Marker in Differential Diagnostics of Autism and Childhood-Onset Schizophrenia.

Introduction: Differential diagnostics of early-onset schizophrenia and autism spectrum disorders (ASD) are a problem of child psychiatry. The prognosis and relevant treatment are to a large degree determined by the correctness of diagnosis. We found earlier that leucocyte DNA of adult schizophrenia patients contained significantly larger copy numbers of ribosomal repeats (rDNA) coding for rRNA, than DNA of mentally healthy controls. Aim: To compare the contents of ribosomal repeats in the leucocyte DNA of children with schizophrenia, children with ASD, and healthy age-matched controls to estimate the possibility of using this genetic trait in the differential diagnostics of the two types of disorders. Patients and methods: Blood samples of patients with infantile autism (A­F84.0 according to ICD-10, N = 75) and with childhood-onset schizophrenia (SZ­F20.8 according to ICD-10, N = 43) were obtained from the Child Psychiatry Department of the Mental Health Research Center. The healthy control blood samples (HC, N = 86) were taken from the Research Centre for Medical Genetics collection. The recruitment of cases was based on the clinical psychopathologic approach. DNA was extracted from blood leukocytes with organic solvents. Nonradioactive quantitative hybridization technique was applied for determining the abundance of ribosomal repeats in the genomes. Statistical processing was performed using StatPlus, Statgraphics and MedCalc. Findings: DNA derived from SZ cases contained 565 ± 163 rDNA copies, which is significantly (p < 10−6) higher than the rDNA content in ASD cases (405 ± 109 copies) and controls (403 ± 86 copies). The HC and A groups did not differ by rDNA copy number (p > 0.4). The genetic trait "rDNA copy number in patient's genome" can potentially be applied as an additional marker in differential diagnostics of childhood-onset schizophrenia and autism spectrum disorders.

NADPH-oxidase 4 gene over-expression in peripheral blood lymphocytes of the schizophrenia patients.

INTRODUCTION: Increased systemic oxidative stress is common in schizophrenia (SZ) patients. NADPH-oxidase 4 (NOX4) is the cell oxidoreductase, catalyzing the hydrogen peroxide formation. Presumably, NOX4 is the main oxidative stress factor in a number of diseases such as cardiovascular diseases and cancer. We hypothesized that NOX4 may be involved in the oxidative stress development caused by the disease in the schizophrenic patients' peripheral blood lymphocytes (PBL). MATERIALS AND METHODS: The SZ group included 100 patients (68 men and 32 women aged 28 ± 11 years). The control group included 60 volunteers (35 men and 25 women aged 25 ± 12 years). Flow cytometry analysis (FCA) was used for DNA damage markers (8-oxodG, É£H2AX), pro- and antiapoptotic proteins (BAX1 and BCL2) and the master-regulator of anti-oxidant response NRF2 detection in the lymphocytes of the untreated SZ patients (N = 100) and the healthy control (HC, N = 60). FCA and RT-qPCR were used for NOX4 and RNANOX4 detection in the lymphocytes. RT-qPCR was used for mtDNA quantitation in peripheral blood mononuclear cells. Cell-free DNA concentration was determined in blood plasma fluorimetrically. RESULTS: 8-oxodG, NOX4, and BCL2 levels in the PBL in the SZ group were higher than those in the HC group (p < 0.001). É£H2AX protein level was increased in the subgroup with high 8-oxodG (p<0.02) levels and decreased in the subgroup with low 8-oxodG (p <0.0001) levels. A positive correlation was found between 8-oxodG, É£H2AX and BAX1 levels in the SZ group (p <10-6). NOX4 level in lymphocytes did not depend on the DNA damage markers values and BAX1 and BCL2 proteins levels. In 15% of PBL of the HC group a small cellular subfraction was found (5-12% of the total lymphocyte pool) with high DNA damage level and elevated BAX1 protein level. The number of such cells was maximal in PBL samples with low NOX4 protein levels. CONCLUSION: Significant NOX4 gene expression was found a in SZ patients' lymphocytes, but the corresponding protein is probably not a cause of the DNA damage.

The Psychoemotional Stress-Induced Changes in the Abundance of SatIII (1q12) and Telomere Repeats, but Not Ribosomal DNA, in Human Leukocytes.

INTRODUCTION: As shown earlier, copy number variations (CNV) in the human satellite III (1q12) fragment (f-SatIII) and the telomere repeat (TR) reflects the cell's response to oxidative stress. The contents of f-SatIII and TR in schizophrenic (SZ) patients were found to be lower than in healthy controls (HC) in previous studies. The major question of this study was: 'What are the f-SatIII and TR CNV dynamic changes in human leukocytes, depending on psychoemotional stress?' MATERIALS AND METHODS: We chose a model of psychoemotional stress experienced by second-year medical students during their exams. Blood samples were taken in stressful conditions (exams) and in a control non-stressful period. Biotinylated probes were used for f-SatIII, rDNA, and TR quantitation in leukocyte DNA by non-radioactive quantitative hybridization in SZ patients (n = 97), HC (n = 97), and medical students (n = 17, n = 42). A flow cytometry analysis was used for the oxidative stress marker (NOX4, 8-oxodG, and γH2AX) detection in the lymphocytes of the three groups. RESULTS: Oxidative stress markers increased significantly in the students' lymphocytes during psychoemotional stress. The TR and f-SatIII, but not the rDNA, contents significantly changed in the DNA isolated from human blood leukocytes. After a restoration period (post-examinational vacations), the f-SatIII content decreased, and the TR content increased. Changes in the blood cells of students during examinational stress were similar to those in SZ patients during an exacerbation of the disease. CONCLUSIONS: Psychoemotional stress in students during exams triggers a universal mechanism of oxidative stress. The oxidative stress causes significant changes in the f-SatIII and TR contents, while the ribosomal repeat content remains stable. A hypothesis is proposed to explain the quantitative polymorphisms of f-SatIII and TR contents under transient (e.g., students' exams) or chronic (in SZ patients) stress. The changes in the f-SatIII and TR copy numbers are non-specific events, irrespective of the source of stress. Thus, our findings suggest that the psychoemotional stress, common in SZ patients and healthy students during exams, but not in a schizophrenia-specific event, was responsible for the changes in the repeat contents that we observed earlier in SZ patients.

Copy Number Variation of Satellite III (1q12) in Patients With Schizophrenia.

Introduction: It was shown that copy number variations (CNVs) of human satellite III (1q12) fragment (f-SatIII) reflects the human cells response to stress of different nature and intensity. Patients with schizophrenia (SZ) experience chronic stress. The major research question: What is the f-SatIII CNVs in human leukocyte as a function of SZ? Materials and Methods: Biotinylated pUC1.77 probe was used for f-SatIII quantitation in leukocyte DNA by the non-radioactive quantitative hybridization for SZ patients (N = 840) and healthy control (HC, N = 401). SZ-sample included four groups. Two groups: first-episode drug-naïve patients [SZ (M-)] and medicated patients [SZ (M+)]. The medical history of these patients did not contain reliable confirmed information about fetal hypoxia and obstetric complications (H/OCs). Two other groups: medicated patients with documented H/OCs [hypoxia group (H-SZ (M+)] and medicated patients with documented absence of H/OCs [non-hypoxia group (NH-SZ (M+)]. The content of f-SatIII was also determined in eight post-mortem brain tissues of one SZ patient. Results: f-SatIII in human leukocyte varies between 5.7 to 44 pg/ng DNA. f-SatIII CNVs in SZ patients depends on the patient's history of H/OCs. f-SatIII CN in NH-SZ (M+)-group was significantly reduced compared to H-SZ (M+)-group and HC-group (p < 10-30). f-SatIII CN in SZ patients negatively correlated with the index reflecting the seriousness of the disease (Positive and Negative Syndrome Scale). Antipsychotic therapy increases f-SatIII CN in the untreated SZ patients with a low content of the repeat and reduces the f-SatIII CN in SZ patients with high content of the repeat. In general, the SZ (M+) and SZ (M-) groups do not differ in the content of f-SatIII, but significantly differ from the HC-group by lower values of the repeat content. f-SatIII CN in the eight regions of the brain of the SZ patient varies significantly. Conclusion: The content of f-SatIII repeat in leukocytes of the most patients with SZ is significantly reduced compared to the HC. Two hypotheses were put forward: (1) the low content of the repeat is a genetic feature of SZ; and/or (2) the genomes of the SZ patients respond to chronic oxidative stress reducing the repeats copies number.

Copy Number Variation of Human Satellite III (1q12) With Aging.

Introduction: Human satellite DNA is organized in long arrays in peri/centromeric heterochromatin. There is little information about satellite copy number variants (CNVs) in aging and replicative cell senescence (RS). Materials and Methods: Biotinylated pUC1.77 probe was used for the satellite III (f-SatIII) quantitation in leukocyte DNA by the non-radioactive quantitative hybridization for 557 subjects between 2 and 91 years old. The effect of RS and genotoxic stress (GS, 4 or 6 µM of K2CrO4) on the f-SatIII CNV was studied on the cultured human skin fibroblast (HSF) lines of five subjects. Results: f-SatIII in leukocyte and HSFs varies between 5.7 and 40 pg/ng of DNA. During RS, the f-SatIII content in HSFs increased. During GS, HSFs may increase or decrease f-SatIII content. Cells with low f-SatIII content have the greatest proliferative potential. F-SatIII CNVs in different individuals belonging to the different generations depend on year of their birth. Children (born in 2005-2015 years) differed significantly from the other age groups by low content and low coefficient of variation of f-SatIII. In the individuals born in 1912-1925 and living in unfavorable social conditions (FWW, the Revolution and the Russian Civil War, SWW), there is a significant disproportion in the content of f-SatIII. The coefficient of variation reaches the maximum values than in individuals born in the period from 1926 to 1975. In the group of people born in 1990-2000 (Chernobyl disaster, the collapse of the Soviet Union, and a sharp decline in the population living standard), again, there is a significant disproportion of individuals in the content of f-SatIII. A similar disproportion was observed in the analysis of a group of individuals born in 1926-1975 who in their youth worked for a long time in high-radioactive environment. Conclusion: In generations that were born and who lived in childhood in a period of severe social perturbations or in conditions of environmental pollution, we found a significant increase in leukocyte DNA f-SatIII variability. It is hypothesized that the change of the f-SatIII content in the blood cells reflects the body response to stress of different nature and intensity.