|Year : 2021 | Volume
| Issue : 3 | Page : 118-125
Comet parameters and plasma 8-Iso-prostaglandins F2α: Common markers of etiopathogenesis in major depression and indicators of antioxidant action of fluoxetine
Rajeev Panwar1, M Sivakumar2
1 Assistant Professor, Department of Anatomy, Shri Sathya Sai Medical College and Research Institute, Chengalpattu, Tamil Nadu, India
2 Professor, Department of Anatomy, JIPMER, Puducherry, India
|Date of Submission||16-Apr-2021|
|Date of Decision||24-Jun-2021|
|Date of Acceptance||28-Jun-2021|
|Date of Web Publication||30-Jul-2021|
Assistant Professor, Department of Anatomy, Shri Sathya Sai Medical College and Research Institute, Ammapettai, Chengalpattu - 603 108, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Major depression can be characterized by isolated episodes of disturbance in psychosomatic functions for a duration of at least two weeks along with intermediate non-symptomatic periods. The factors playing important role in the etiology of major depression were family history, genetic factors, decreased levels of serotonin and norepinephrine; increased levels of interleukins or any other medical illness having an increased cytokine production. The pathogenesis of major depression involves oxidative stress, which consists of synthesis of free radicals causing damage to nucleic acids, lipids, carbohydrates, and proteins present in the cells. Oxidative stress – induced DNA damage consists of numerous types of lesions which can be assessed by the comet assay method. Prostaglandin F2-alpha is produced because of oxidative stress-induced peroxidation of cell membrane lipids and has elevated plasma levels in patients with major depression. Fluoxetine is the drug of choice for major depression and found to have antioxidant properties. The present study was done to assess the correlation between plasma 8-iso-PGF2α levels and the comet parameters, namely comet length (CL), head diameter (HD), % of DNA in head (%DNA Head), tail length (TL) and % of DNA in tail (%DNA Tail), before starting and after finishing the eight-week fluoxetine therapy. Methodology: The prospective clinical study was conducted in the Department of Anatomy in collaboration at, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. The study group consisted of drug naïve, newly diagnosed major depression patients belonging to age-group of ≥ 18 to 50 years and followed up after eight weeks of fluoxetine therapy. Results: The correlation was found to be positive between the levels of 8-iso-PGF2α in the plasma and all the comet parameters except %DNA Tail, which showed a negative (inverse) relationship with the levels of 8-iso-PGF2α in the plasma. Conclusion: The current study suggested the role of oxidative stress in causing DNA damage and lipid peroxidation in major depression patients and the antioxidant role of fluoxetine in causing decrease in the levels of parameters of oxidative stress and subsequent DNA repair.
Keywords: 8-Iso-Prostaglandins F2α, comet assay, DNA damage, fluoxetine, major depression, oxidative stress
|How to cite this article:|
Panwar R, Sivakumar M. Comet parameters and plasma 8-Iso-prostaglandins F2α: Common markers of etiopathogenesis in major depression and indicators of antioxidant action of fluoxetine. Natl J Clin Anat 2021;10:118-25
|How to cite this URL:|
Panwar R, Sivakumar M. Comet parameters and plasma 8-Iso-prostaglandins F2α: Common markers of etiopathogenesis in major depression and indicators of antioxidant action of fluoxetine. Natl J Clin Anat [serial online] 2021 [cited 2021 Oct 22];10:118-25. Available from: http://www.njca.info/text.asp?2021/10/3/118/322805
| Introduction|| |
Major depression can be defined as isolated spells of changes in concern, intellect, psychosomatic functions lasting for at least 2 weeks along with intermediate symptom-free periods. Women, especially belonging to 45–64 years of age group, widowed or divorced, had a higher lifetime risk of major depression. Higher prevalence of major depression among females was suggested in a study on urban population in South India, with depressed mood being the most regularly present symptom. The factors playing an important role in the etiology of major depression were family history, genetic factors, decreased levels of serotonin and norepinephrine, increased levels of interleukin-6 (IL-6), C-reactive protein, IL-1s946;, medical illness due to any reason, or any other condition having an increased cytokine production.,,,
The role of decrease in the levels of catecholamines and serotonin and increase in the levels of macrophage monokines such as IL-1, interferon alpha (INF-α), and tumor necrosis factor (TNF), as well as monocytes and T-lymphocytes in the pathogenesis of major depression has been described in the past.,,,, The changes in the levels of catecholamines, serotonin, and monokines lead to activation of inflammatory response system (IRS).,,, Activation of IRS causes hypothalamic–pituitary–adrenal axis hyperactivity, hypothalamic–pituitary–thyroid axis alterations, and dysfunctions in serotonin or catecholamines turnover, which ultimately manifests as major depression., Thus, oxidative stress plays a significant role in the development of major depression, and it is characterized by synthesis of free radicals, resulting in damage to nucleic acids, lipids, carbohydrates, and proteins present in the cells.
Oxidative stress-induced DNA damage consists of numerous types of lesions which can be assessed by the comet assay method., During comet assay, single-cell gel electrophoresis (SCGE) technique is utilized for estimation of the migration of the damaged DNA. Comet assay suitable for alkaline medium was demonstrated by Singh et al., which was later simplified by Collins et al.,
The breaks in the DNA strand resulted in the liberation of supercoils, leading to migration of the fragmented DNA toward the anode, giving a comet-like appearance. The comet can be visualized under the microscope, and the captured images can be utilized for assessment of comet parameters using comet software. Among all the comet parameters, TL and %DNA Tail were believed to be the best indicators of DNA damage.,
Oxidative stress-induced peroxidation of cell membrane lipids causes significant increase in the plasma levels of 8-iso-prostaglandins F2-alpha (8-iso-PGF2α) in major depression patients. The assessment of levels of 8-iso-PGF2α in the plasma or urine was considered as highly precise technique for determining the presence and degree of oxidative stress. Immunoassay kits containing antibodies against 8-iso-PGF2α have been used for the estimation of levels of 8-iso-PGF2α in the plasma.
The most preferred drug for major depression having minimal or nonsignificant effects on the cardiac function is fluoxetine. Fluoxetine inhibits presynaptic receptor mediated re-uptake and subsequent degradation of serotonin by monoamine oxidase and causes continuous activation of postsynaptic receptors by serotonin. It has the ability to restore the activity of glutathione reductase leading to increase in cellular levels of glutathione, and it increases the levels of glucose and uric acid in brain, all of which protect against highly damaging hydroxyl radicals that react with and damage most cellular targets, including lipids, proteins, and DNA. Thus, fluoxetine reduces oxidative stress-induced damage and causes restoration of antioxidant function along with its therapeutic effect.
There could be an association between the levels of comet parameters and plasma 8-iso-PGF2α as both, DNA damage and lipid-peroxidation, are the consequence of oxidative stress. Grindel et al. assessed the levels of parameters of oxidative stress, DNA damage, and repair of the damaged DNA in female patients with diabetes mellitus type II. However, the association between comet parameters and 8-iso-PGF2α was not described. After extensive search of the literature, no study was found which described analysis of association between the levels of comet parameters and the plasma levels of 8-iso-PGF2α among major depression patients before starting and after treatment with fluoxetine.
In the present study, correlation was analyzed between plasma 8-iso-PGF2α levels and the comet parameters, before starting and after completing the 8-week fluoxetine therapy.
| Materials and Methods|| |
The prospective clinical study was conducted from October 2016 to March 2018 in the Department of Anatomy in collaboration with the Departments of Psychiatry and Biochemistry at Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, after getting clearance from the Postgraduate Research Monitoring Committee and Institute Ethics Committee (Ethical Committee Clearance certificate number JIP/IEC/2016/28/941 dated October 22, 2016).
The study group consisted of drug naïve, major depression patients newly diagnosed, as per fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM 5) criteria, belonging to age group of ≥18–50 years and followed up after 8 weeks of fluoxetine therapy. All the patients received 20 mg fluoxetine single dose at night for 8 weeks. All the patients were drug naïve and newly diagnosed with major depression; hence, there was not any difference in the drug dose schedule. Pregnant women, patients with medical illnesses such as diabetes mellitus/hypertension/cancer/genetic disorder or any other long-standing illnesses, and patients on antidepressant medications or with any other documented psychiatric illness were excluded from the study. While taking the history in the outpatient department, the patients with a history of smoking/alcohol intake/sedentary lifestyle, etc., were excluded from the study.
Open Epi program 9 open sources Epidemiology, Statistics for Public Health version 3.01 formula for comparing two means was used for estimation of the sample size. On assuming an alpha error of 0.05, power 90%, and 10% dropouts, 80 newly diagnosed major depression patients were recruited for the study. Out of total 80 patients, 47 were females (58.75%) and 33 were males (41.75).
The study consisted of an analysis of correlation between two different indicators of oxidative stress, namely (i) comet parameters, markers of DNA damage, using comet assay technique, and (ii) plasma 8-iso-PGF2α, a marker of lipid peroxidation, using enzyme-linked immunosorbent assay (ELISA) method.
Ástling and Johansson used SCGE technique while doing the comet assay for the estimation of DNA damage. The procedure was modified by Singh et al., to a more convenient, alkaline comet assay. The alkaline comet assay method standardized by Nandhakumar et al. was used in the current study for the assessment of DNA damage., The principle of alkaline comet assay involved the movement of the negatively charged damaged DNA fragments toward the anode during electrophoresis and appearing as comet-like tail under the microscope.
In the current study, the alkaline comet assay was performed in a 3-day protocol and consisted of blood collection to cell lysis on day 1, electrophoresis and cell fixation on day 2, and staining and comet scoring on day 3.
2 ml of peripheral venous blood and 2 ml of histopaque were taken in test tube and centrifuged for 30 min, which resulted in separation of plasma from three distinct layers. Among the three layers, red blood corpuscles (RBCs) were settled at the bottom, histopaque was in the middle, and a buffy coat of lymphocytes was at the top. The lymphocytes were mixed with low melting agar (LMA) and layered on a glass slide precoated normal melting agar (NMA). The LMA and NMA were used at 37°C for preparing the slides and were dried at 4°C in a refrigerator for 10–15 min. This was followed by coating with a third layer of LMA; thus, lymphocytes were sandwiched between two layers of agarose and kept overnight at 4°C in the lysis solution for lysis of the cell membrane of the lymphocytes to expose the nucleoids.
Lysis stock solution is prepared by dissolving 146.1 g NaCl, 37.2 g disodium ethylene diamine tetra-acetic acid (Na2EDTA), and trisaminomethane (TRIS) in 700 ml double distilled (dd) water followed by adding 12 g sodium hydroxide (NaOH) pallets and stirring. Then, 10 g sodium lauryl sarcosinate or 1 g sodium dodecyl sulfate was added which is followed by stirring and setting the pH to 10. Final volume is adjusted to 890 ml, filtered, and kept at room temperature.
Lysis solution (working solution) is prepared by adding 0.1 ml of Triton-X to 9.9 ml of dd water and mixed well followed by adding the 1.6 ml from this solution to 98.4 ml of lysis stock solution, and the resultant lysis working solution is kept in the refrigerator.
The slides were subjected to electrophoresis without current for 20 min for unwinding of DNA followed by passage of the current for 20 min, resulting in migration of negatively charged damaged DNA fragments toward the anode. Electrophoresis solution (stock solution) consisted of electrophoresis solution-I which was prepared by dissolving 200 g NaOH in 500 ml dd water and electrophoresis solution-II, which was prepared by adding 14.89 g Na2EDTA in 200 ml dd water and then the pH was adjusted to 10.
Electrophoresis working solution was prepared by mixing 30 ml of electrophoresis stock solution-I with 5 ml of electrophoresis stock solution-II, and then, the volume was adjusted to 1000 ml by adding chilled dd water. The standard electrophoresis protocol developed by Singh et al. consisted of 25 V and 300 mA. Further, electrophoresis can be conducted at temperature ranging from 5°C to room temperature; the use of a lower temperature is thought to provide increased reproducibility. This is followed by neutralization with TRIS buffer, washing with distilled water, and overnight drying. The neutralization buffer solution was prepared by dissolving 48.5 g of TRIS in 800 ml of dd water, and then, pH was adjusted to 7.5 by adding concentrated hydrochloric acid and final volume was adjusted to 1000 ml.
Neutralized-dried slides were fixed in fixative solution, washed with dd water, and dried at room temperature followed by staining with silver nitrate solution. The stained slides were visualized under bright field microscope for capturing the images which were analyzed by using TriTek CometScore™ Freeware v1.5 software. 40–50 randomly selected cells per patient were seen under the microscope. The chosen comets were representing the entire gel without any predisposition. The comets present in the edges, air bubbles, and the overlaps were rejected.
For assessment of DNA damage, levels of comet length (CL), head diameter (HD), percentage of DNA in the head (%DNA Head), tail length (TL), and percentage of DNA in the tail (%DNA Tail) were analyzed in the samples taken before starting and after completion of 8-week fluoxetine therapy.
CL of the comet is the total amount of DNA present in one lymphocyte, the head of the comet contains the undispersed DNA, i.e., the undamaged DNA, the tail of the comet has the DNA, which is fragmented and hence has more negatively charged molecules and moves more toward the anode, i.e., the damaged DNA [Figure 1].
Estimation of 8-iso-prostaglandins F2-alpha
The top layer of centrifuge consisting of plasma was isolated from 80 major depression patients before starting and after completion of the 8-week therapy with fluoxetine, stored at −80°C temperature, and utilized for the estimation of 8-iso-PGF2α by the colorimetric method using 8-iso-PGF2α ELISA kit of the Bioassay Technology Laboratory. The ELISA kit had Cat. No E1179Ra, standard curve range: 0.5–150 ng/L, sensitivity: 0.22 ng/L, size: 96 wells, and temperature required for storage of the reagents at 2°C–8°C.
The information on continuous parameters such as age, comet parameters, and plasma F2-isoprostane (isoPs) was expressed as mean ± standard deviation or median with interquartile range. The comparison of the comet parameters and plasma F2-isoPs between the groups was carried by utilizing Student's paired t-test/Wilcoxon signed-rank sum test, whichever was appropriate given the number of groups (at least two) and distribution of data (normal/nonnormal). Among all the comet parameters, only HD followed a normal distribution, remaining all other comet parameters, i.e., CL, %DNA Head, TL, and %DNA Tail, followed nonnormal distribution. The plasma 8-iso-PGF2α levels in major depression cases before the treatment followed the Gaussian pattern (normal distribution), while in the cases after the treatment, there was non-Gaussian pattern (nonnormal distribution).
For analyzing the correlation between plasma F2-isoPs levels and HD before the treatment, Pearson's correlation test was done, while for analyzing correlation between plasma F2-isoPs levels and rest all other comet parameters, Spearman's rho correlation test was done.
| Results|| |
On comparing the levels of comet parameters before starting and after completion of the treatment, it was found that there was a reduction in the levels of all the parameters suggesting decrease or arrest of DNA damage due to fluoxetine but increase in the levels of % DNA Head, suggesting DNA damage repair action of fluoxetine. All the changes were statistically significant except changes in the levels of head diameter [Table 1]. The levels of plasma F2-isoPs were also reduced after 8-week therapy with fluoxetine, suggesting its antioxidant action, and the differences were statistically significant [Table 2].
|Table 1: Summary of comet parameters in major depression patients before starting and after completion of the 8-week therapy with fluoxetine|
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|Table 2: Comparison of plasma F2-isoprostane levels in major depression patients before starting and after completion of the 8-week therapy with fluoxetine|
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Gender-wise comparison of comet parameters among major depression patients before starting and after completion of the 8-week therapy with fluoxetine was done, and it was found that the total CL, HD, TL, and %DNA Tail were decreased after the treatment and there was increase in %DNA Head after the treatment among both male and female major depression patients. All the changes were statistically significant [Table 3].
|Table 3: Comparison of comet parameters among female and male major depression patients before starting and after completion of the eight-week therapy with fluoxetine|
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Among all the cases, statistically significant correlation was found only between plasma 8-iso-PGF2α levels and HD before starting the treatment and between plasma 8-iso-PGF2α levels and CL after completion of the treatment (with P = 0.020 and 0.038, respectively), and in rest all the cases either before starting or after completing the treatment, the correlation was statistically nonsignificant (P > 0.05) [Table 4].
|Table 4: Correlation of F2-isoprostane levels with comet parameters in major depression patients before starting and after completion of the 8-week therapy with fluoxetine|
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On performing the simple correlation analysis along with the scatter plot, changes in the levels of plasma 8-iso-PGF2α among the cases before starting and after completion of 8-week fluoxetine therapy were showing positive relationship with CL [Figure 2], HD [Figure 3], %DNA Head [Figure 4], and TL [Figure 5] and negative relationship with %DNA Tail [Figure 6].
|Figure 2: Linear correlation between 8-iso-prostaglandins F2-alpha and total comet length: (a) Before treatment. (b) After treatment|
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|Figure 3: Linear correlation between 8-iso-prostaglandins F2-alpha and head diameter: (a) Before treatment. (b) After treatment|
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|Figure 4: Linear correlation between 8-iso-prostaglandins F2-alpha and percentage of DNA in head: (a) Before treatment. (b) After treatment|
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|Figure 5: Linear correlation between 8-iso-prostaglandins F2-alpha and tail length: (a) Before treatment. (b) After treatment|
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|Figure 6: Linear correlation between 8-iso-prostaglandins F2-alpha and percentage of DNA in tail: (a) Before treatment. (b) After treatment|
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| Discussion|| |
Major depression is characterized by the presence of symptoms categorized by criterion “A–C” as suggested by DSM 5. Depression was more prevalent among Indian females, and higher morbidity and mortality was due to higher incidences of attempted suicides.,
Complete or partial deficiency of catecholamines, specifically norepinephrine, serotonin, or 5-hydroxytryptamine, and increase in the levels of macrophage monokines such as IL-1, INF-α, and TNF have been suggested as stressor responsible for activation of IRS, which play a significant role in the development of depression.,,,, Maes et al. suggested the role of inflammatory, oxidative, and nitrosative pathways, leading to increase in the levels of cytokines and neurotoxic metabolite tryptophan catabolites along the indoleamine oxidase and reduction in plasma levels of tryptophan. Reduction in the levels of tryptophan manifested as behavioral changes, neurodegenerative changes, and immunoglobulin M mediated autoimmmunological reaction against the lipid membrane components.,, The free radicals such as reactive oxygen species and reactive nitrogen species have highly reactive oxidizing properties, and their levels were kept under check by antioxidants. Any disparity between levels of oxidants and antioxidants resulting in shifting of equilibrium toward oxidants is known as oxidative stress.
Oxidative stress causes various types of structural lesions in DNA such as apurinic/apyrimidinic/baseless sites, cyclobutane-type pyrimidine dimer, chemical alteration in small bases, distortion in helix structure, and breaks in single and double strands in nuclear or mitochondrial DNA., Czarny et al. proposed comet assay method for the estimation of elevated levels of breakage in DNA strands and alkali-labile sites in the recurrent depressive disorder patients. Singh et al. proposed modified protocol for comet assay, suitable for alkaline conditions which was later simplified by Collins et al., by suggesting a single layer of agarose gel for cell embedding on a plain slide precoated with agarose., In the comet structure, the undamaged DNA can be indicated by the head of the comet and the damaged DNA is known as the tail of the comet; hence, the amount of DNA in the tail is directly proportional to the degree of DNA damage. Any increase in either the total CL or TL will indicate the DNA damage and increase in the HD will indicate DNA damage repair. The degree of DNA strand breakage can be identified by estimating % of DNA head & % of DNA tail with the help of commercially available software., isoPs are product of lipid peroxidation of cell membrane phospholipids, existing as various isomers such as F2-isoPs, F3-isoPs, and F4-isoPs.,,
The role of fluoxetine in influencing the antioxidant defenses and peroxidation of the lipids was described by Galecki et al. by estimating the levels of superoxide dismutase, catalase, malondialdehyde, and Hamilton depression rating scale in major depression patients. Lindqvist et al. analyzed different parameters of oxidative stress and found the differences between the levels of parameters such as TNF-α, IL-6, 8-iso-PGF2α, and 8-OHdG in the individuals having major depression and the healthy ones to be statistically significant. Furthermore, the levels of markers of oxidative stress (e.g., 8-iso-PGF2α, 8-OHdG) and inflammation markers (e.g., IL-6) were influenced by antidepressant treatment. Raza et al. described the role of DNA damage in various psychiatric disorders such as major depressive disorders, schizophrenia, and bipolar disorders, but none of the study assessed the levels of comet parameters in the major depression patients before starting and after completion of treatment with fluoxetine.
In the present study, after 8-week fluoxetine therapy, there was reduction in the levels of comet parameters, suggestive of oxidative stress-induced DNA damage such as HD, CL, TL, and %T, but increase in the levels of comet parameter, suggestive of DNA repair such as % H. Furthermore, levels of 8-iso-PGF2α, a marker of oxidative stress-induced lipid peroxidation, were also reduced suggesting antioxidative properties of fluoxetine. A positive relationship was found between the levels of 8-iso-PGF2α in the plasma and all the comet parameters except %T, in the cases, before starting and after concluding the treatment with fluoxetine.
| Conclusion|| |
It is concluded that oxidative stress plays significant role in etiopathogenesis of major depression via various common pathways, which is explained by positive association between plasma 8-iso-PGF2α levels and all the comet parameters, in the cases, before starting the treatment, as both are indicators of oxidative stress. In addition, there is a positive association among plasma 8-iso-PGF2α levels and all the comet parameters in the cases after completion of 8-week fluoxetine therapy, suggesting that fluoxetine has antioxidant properties along with its therapeutic role since markers of oxidative stress namely plasma 8-iso-PGF2α levels and comet parameters were reduced after the treatment.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]