Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients

https://doi.org/10.1016/j.jpsychires.2016.12.006Get rights and content

Highlights

  • Cell-free DNA quantity (cfDNA) was determined for the first time in plasma of SZ patients.

  • A high level of 8-oxodG was found in cell-free DNA of SZ patients.

  • A high level of 8-oxodG (FL1-8-oxodG) was found in SZ lymphocytes.

  • The ratios cfDNA/FL1-8-oxodG are strongly lowered for one-third SZ patients.

  • One-third of SZ patients have impaired apoptosis.

Abstract

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.

Introduction

Oxidative DNA damage has been proposed as a causer in the most recent hypotheses concerning the pathophysiology of SZ (Raza et al., 2016, Sertan Copoglu et al., 2015). The endogenous source of damage are reactive oxygen species (ROS) emerging as by-products from oxygen metabolism. Elevated ROS levels and declined antioxidant statuses in the brain and peripheral tissues of the patients with SZ have been reported (Emiliani et al., 2014, Boskovic et al., 2011, Smaga et al., 2015). DNA damage triggers a number of cellular responses including arrest of the cell cycle progression in order to facilitate DNA repair or eliminate damaged cells through apoptosis (Rouse and Jackson, 2002, Uttara et al., 2009). 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents major oxidative DNA damage products. An elevated level of 8-oxodG in the blood and urine has been reported in SZ patients (Sertan Copoglu et al., 2015, Jorgensen et al., 2013). It should be noted that blood and urine levels of extracellular nucleoside 8-oxodG depend on many parameters involving the individual peculiarities of the metabolic processes and internal organ functional status, therefore this parameter is just an approximate indicator of the real DNA oxidation in the cell. Post mortem studies have revealed a higher level of 8-oxodG in the midbrain, caudate putamen, and hippocampus in SZ patients compared to the controls (Cardozo-Pelaez et al., 2000, Nishioka and Arnold, 2004). However, there is no reports on the measurements in vivo of 8-oxodG content in the cells of SZ patients.

It is known that cells with the highest degree of DNA damage are eliminated via apoptosis. Neuronal apoptosis has been hypothesized to contribute to the pathophysiology of SZ at early stages of the pathogenesis (Jarskog, 2006, Cabungcal et al., 2013). Also there is a growing body of evidence obtained in post mortem studies that apoptotic impairments are involved in the pathophysiology of SZ (Jarskog et al., 2004, Benes et al., 2003, Benes et al., 2006, Catts et al., 2006). However, there is only one article, in which the serum level of apoptosis marker in SZ patients was detected (Beyazyüz et al., 2016).

Earlier, we developed an approach to estimate cell death intensity in the body of subjects with chronic oxidative stress (Korzeneva et al., 2015). The approach is based on comparing the levels of DNA damage in lymphocytes and the concentration of cell-free circulating DNA. The term “cell-free circulating DNA” (cfDNA) was coined for DNA fragments that could be collected from plasma, serum, or other bodily fluids. CfDNA circulates throughout the blood stream of both healthy people and patients and got recognition as a promising biomarker for noninvasive diagnostics and monitoring of many various diseases (Fleischhacker and Schmidt, 2007). To the best of our knowledge, no published studies have examined cfDNA in patients with psychiatric disorders. The main sources of cfDNA are the dying cells (Jahr et al., 2001, van derVaart and Pretorius, 2008). So, cfDNA concentration is an indicator of the level of cell death in the body. The analysis of cfDNA oxidation is important in order to predict its biological action on the cells of the organism. It was previously shown that oxidized cfDNA is one of the considerable factors of stress signaling that affects various cell types (Ermakov et al., 2013).

The aforementioned approach has enabled us to differentiate the response of irradiation exposed male for the chronic oxidative stress (Korzeneva et al., 2015). Some of the male developed a prominent adaptive response aimed at increasing the cell survivability, including cells with damaged DNA. The results of this kind of response resemble the published post mortem data for the brain of SZ patients: a decline in apoptosis at the background of high DNA damage degree (Benes et al., 2003, Benes et al., 2006).

We proposed that a combined analysis (1) of DNA damage in the blood lymphocytes and (2) plasma cfDNA concentration and oxidation would allow dividing the patients into subgroups, which are distinct by the general level of cell death in the body. To test this hypothesis, we analyzed the following parameters in SZ patients with acute psychotic disorders: (1) 8-oxodG content in the blood lymphocytes; (2) cfDNA concentration in the blood plasma, and (3) 8-oxodG content in the cfDNA. Joint assay of these indices allowed us to divide the SZ patients into subgroups, which are distinct by the lymphocyte DNA damage degree and the level of the cell death.

Section snippets

Subjects

The investigation was carried out in accordance with the latest version of the Declaration of Helsinki and approved by the Regional Ethics Committee of RCMG (approval #5). All participants signed an informed written consent to participate after the nature of the procedures had been fully explained. 83 male patients with acute psychotic disorders were recruited from general psychiatric unit for treatment of acute forms of mental disorders between December 2015 and August 2016. 58 patients with

Results

83 male patients with acute psychosis were enrolled in this study: 58 paranoid SZ patients (SZ), 11 patients with schizophreniform psychotic disorder (SDP), 14 patients with alcohol-induced psychotic disorder (AIPD). Venous blood was sampled immediately after hospitalization before therapy. The control group consisted of 30 healthy male subjects.

Discussion

SZ is increasingly considered as a systemic disorder, which is associated with biochemical disturbances not only in the CNS/brain cells (Kirkpatrick, 2009). Works conducted by a series of authors suggest that an examination of any cells available at schizophrenia (fibroblasts (Mahadik and Mukherjee, 1996), lymphocytes (Muraleedharan et al., 2015, Catts et al., 2006), lymphoblasts (Catts et al., 2012)) and easily accessible biological fluids (urine (Miyaoka et al., 2016), peripheral blood

Role of funding source

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare that they have no conflict of interest.

Contributors

Ershova, Jestkova and Chestkov provides clinical samples, participated designing and carrying out most of the experimental procedures. Veiko and Kostyuk participated performing the statistical analyses and the interpretation of results, and writing the first draft of the manuscript. Izevskaya and Kutsev participated in the coordination, conceiving and design of the study and helped in the drafting of the manuscript. Porokhovnik translated the article into English and helped in the drafting of

Acknowledgments

The authors thank Roman Veiko for the software for MLE and “Imager6”.

References (51)

  • S. Balusu et al.

    Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles

    EMBO Mol. Med.

    (2016)
  • F.M. Benes et al.

    The expression of proapoptosis genes is increased in bipolar disorder but not in schizophrenia

    Mol. Psychiatry

    (2006)
  • F.M. Benes et al.

    DNA fragmentation decreased in schizophrenia but not in bipolar disorder

    Arch. General Psychiatry

    (2003)
  • M. Beyazyüz et al.

    Increased serum levels of apoptosis in deficit syndrome schizophrenia patients: a preliminary study

    Neuropsychiatr. Dis. Treat.

    (2016)
  • M. Boskovic et al.

    Oxidative stress in schizophrenia

    Curr. Neuropharmacol.

    (2011)
  • J.H. Cabungcal et al.

    Early-life insults impair parvalbumin interneurons via oxidative stress: reversal by N-acetylcysteine

    Biol. Psychiatry

    (2013)
  • F. Cardozo-Pelaez et al.

    DNA damage, repair, and antioxidant systems in brain regions: a correlative study

    Free Radic. Biol. Med.

    (2000)
  • V.S. Catts et al.

    Evidence of aberrant DNA damage response signaling but normal rates of DNA repair in dividing lymphoblasts from patients with schizophrenia

    World J. Biol. Psychiatry

    (2012)
  • V.S. Catts et al.

    Apoptosis and schizophrenia: a pilot study based on dermal fibroblast cell lines

    Schizophr. Res.

    (2006)
  • M. Chakravadhanula et al.

    Detection of an atypical teratoid rhabdoid brain tumor gene deletion in circulating blood using next-generation sequencing

    J. Child Neurol.

    (2014)
  • I.D. Connolly et al.

    The “Liquid Biopsy”: the role of circulating DNA and RNA in central nervous system tumors

    Curr. Neurol. Neurosci. Rep.

    (2016)
  • L.V. Efremova et al.

    Accumulating Fragments of Extracellular DNA (EcDNA) Influence Rat Primary Cerebellum Granule Cell Culture

    (2010)
  • F.E. Emiliani et al.

    Oxidative stress and schizophrenia: recent breakthroughs from an old story

    Curr. Opin. Psychiatry

    (2014)
  • A.V. Ermakov et al.

    Oxidized extracellular DNA as a stress signal in human cells

    Oxid. Med. Cell. Longev.

    (2013)
  • M. Fleischhacker et al.

    Circulating nucleic acids (CNAs) and cancer—a survey

    Biochim. Biophys. Acta

    (2007)
  • K. Glebova et al.

    Oxidized extracellular DNA as a stress signal that may modify response to anticancer therapy

    Cancer Lett.

    (2015)
  • K.V. Glebova et al.

    Oxidative modifications of DNA alter its biological action on rat neurons

    J. Nucleic Acids Investig.

    (2011)
  • K.V. Glebova et al.

    Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease

    Bull. Exp. Biol. Med.

    (2014)
  • J. Guz et al.

    The relationship between 8-oxo-7,8-dihydro-2’-deoxyguanosine level and extent of cytosine methylation in leukocytes DNA of healthy subjects and in patients with colon adenomas and carcinomas

    Mutat. Res.

    (2008)
  • A. Ilhan-Mutlu et al.

    Circulating biomarkers of CNS tumors: an update

    Biomarkers Med.

    (2013)
  • S. Jahr et al.

    DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells

    Cancer Res.

    (2001)
  • L.F. Jarskog et al.

    Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation

    Am. J. Psychiatry

    (2004)
  • L.F. Jarskog

    Apoptosis in schizophrenia: pathophysiologic and therapeutic considerations

    Curr. Opin. Psychiatry

    (2006)
  • A. Jorgensen et al.

    Increased systemic oxidatively generated DNA and RNA damage in schizophrenia

    Psychiatry Res.

    (2013)
  • I.L. Konorova et al.

    Emotional stress in rats changes concentration and composition of extracellular DNA circulating in blood plasma under normal conditions and in cerebral ischemia

    Bull. Exp. Biol. Med.

    (2012)
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