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Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay
Journal of Psychiatric Research 68 (2015) 47e53
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Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay Aparna Muraleedharan a, Vikas Menon b, *, Ravi Philip Rajkumar b, Parkash Chand a a b
Department of Anatomy, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Dhanvantary Nagar, Puducherry, 605006, India Department of Psychiatry, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Dhanvantary Nagar, Puducherry, 605006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2015 Received in revised form 10 May 2015 Accepted 12 May 2015
The etiology of schizophrenia continues to be confounding and elusive. Some knowledge gaps exist in the neurodegenerative theory of schizophrenia. Oxidative DNA damage and repair deficits are relevant to the mechanisms of neurodegeneration but have not been studied in drug naïve schizophrenia. The present study used the comet assay technique to study the extent of DNA damage in circulating peripheral lymphocytes of patients with drug naïve schizophrenia (n ¼ 40) along with an age and gender matched control group (n ¼ 40). We also assessed the DNA repair efficiency in cases following incubation in a nutrient medium. All the assayed comet parameters demonstrated significantly greater baseline DNA damage in cases in comparison to the controls except for head diameter (p < 0.001 for all significant results, p ¼ 0.32 for head diameter). Gender, age and duration of illness (p ¼ 0.21, 0.69 and 0.12 respectively for tail length) did not influence any of the parameters significantly. Significant decrease was noted in the comet tail length and percentage of DNA in comet tail (p < 0.001 for both) in cases following incubation suggesting that the DNA repair machinery was preserved. No difference in DNA repair efficiency was noted between the genders (p ¼ 0.23 for tail length). Our findings confirm the presence of significant baseline DNA damage in schizophrenia even prior to the initiation of anti-psychotic treatment. Additionally, intact genomic repair efficiency was noted in this group as a whole. These results provide some evidence for oxidative DNA damage as molecular link underpinning neurodegeneration in drug naïve schizophrenia. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Schizophrenia Drug naive DNA damage Genetic repair Neurodegeneration
1. Introduction Schizophrenia is a chronic, debilitating psychiatric disorder whose etiology has been challenging to decipher. There are, broadly speaking, three hypothesis put forth to explain the etiopathogenesis of this condition. They are the neurodevelopmental (Fatemi and Folsom, 2009), neurodegenerative (Christopoulos et al., 2006) and the progressive neurodevelopmental model (Woods, 1998). The term neurodegeneration refers to chronic, progressive disorders of the nervous system characterized by continuing neuronal loss. The prototype examples of such disorders are Alzheimer's et al., 2006). Numerous disease and Huntington's disease (Coppede
* Corresponding author. Dept. of Psychiatry, JIPMER, Dhanvantary Nagar, Puducherry 605006, India. þ91 9894410296 (mobile); fax: þ91 413 2272067. E-mail address: [email protected] (V. Menon). http://dx.doi.org/10.1016/j.jpsychires.2015.05.018 0022-3956/© 2015 Elsevier Ltd. All rights reserved.
lines of evidence support the neurodegenerative theory of schizophrenia. Longitudinal neuroimaging studies have been able to demonstrate progressive changes in the size and function of frontal and temporal lobes following illness onset lending credence to this theory (Shenton et al., 2001; Kasai et al., 2003; Buckley, 2005). Oxidative stress is a hallmark of any neurodegenerative process and is also believed to be involved in the pathophysiology of schizophrenia. Evidence has accumulated regarding the decreased levels of endogenous anti-oxidants such as superoxide dismutase, glutathione and catalase in people with schizophrenia (Fendri et al., 2006; Dadheech et al., 2008; Tsai et al., 2013). On a conflicting note, gliosis which is another feature of neurodegeneration has been notably absent in histopathological models of schizophrenia indicative of knowledge gaps about this hypothesis (Harrison 1999; Schnieder and Dwork, 2011). Deoxyribonucleic Acid (DNA) damage can be defined as any alteration in DNA that induces a change in its function or modifies
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its coding properties (Lindahl, 1993; Rao, 1993). Compelling evidence, from animal and invitro models, suggest that DNA strand breaks are early antecedents of both apoptoptic and necrotic forms of neuronal cell death (Liu and Martin, 2001; Martin et al., 2006). Studies using cultured embryonic mouse cortical neurons have demonstrated the rapid accumulation of increasing DNA-single strand breaks (DNA-SSBs) with increasing levels of oxidative stress prior to eventual cell death. Furthermore, the timing of these genomic strand breaks in neurodegenerative disease models suggests that they are forerunners of a p53-driven apoptotic cascade in the cerebral cortex (Martin et al., 2009). Interestingly, authors who investigated mechanisms of motor neuron (MN) degeneration using transgenic mice expressing human mutant superoxide dismutase-1 (mSOD1) identified novel patterns of MN degeneration that differed from apoptosis structurally and biochemically. They concluded that there is a role for oxidative stress in the process of motor neuron degeneration induced by mSOD1 (Martin et al., 2007). Based on many of these findings, researchers have posited that oxidative stress induced genomic damage is relevant to the mechanisms resulting in neurodegeneration (Martin, 2008). Oxidative stress is thought to activate DNA damage pathways and play a critical role in activating telomere erosion, a key molecular correlate of the aging process (Sahin and DePinho, 2010). Some authors have suggested that this chronic state of oxidative stress on nucleic acids may underpin some of the clinical indicators of early aging in schizophrenia such as mildly accelerated cognitive decline (Jorgensen et al., 2013). DNA repair mechanisms have also been noted to be defective in many neurodegenerative conditions (Kidson and Chen, 1986). In a recent review that specifically focused on consequences of defective DNA repair mechanisms in non-proliferating cells such as neurons, the authors concluded that inefficient DNA repair has a robust link with aging and manifestations of classic neurodegenerative conditions such as Alzheimer's disease (Madabhushi et al., 2014). The evidence for neurodegeneration in schizophrenia remains sketchy. Previous investigators who studied urinary markers of oxidative DNA damage using ultra performance liquid chromatography with tandem mass spectroscopy concluded that there was a statistically significant increase in the excretion of these markers in schizophrenia patients when compared to matched controls (Jorgensen et al., 2013). Evidence for increased levels of oxidative DNA damage markers in schizophrenia have also come from studies on post mortem hippocampi in elderly patients with poor clinical outcomes (Nishioka and Arnold, 2004). By contrast, DNA repair response in schizophrenia has received comparatively less research attention. In a study using immortalized lymphoblasts from schizophrenia patients and healthy controls, the authors investigated the issue of DNA repair by studying temporal change in markers of DNA-DSBs following irradiation and concluded that there was no difference in rates of DNA repair between the two groups (Catts et al., 2012). Single cell gel electrophoresis or “comet” assay is regarded as a sensitive method for estimating the major types of DNA damage lesions in eukaryotic cells (Tice et al., 2000). Few studies have used this technique to measure levels of DNA damage and repair in schizophrenia. Further, most of the available data are from chronic schizophrenia patients who carry a lot of potential confounders that may contaminate these observations, particularly, antipsychotic treatment which has been shown to negatively influence genotoxic parameters in pre-clinical and clinical research (Polischouk et al., 2007; Picada et al., 2011). Hence, we undertook the present study with the twin objectives of assessing DNA damage and repair efficiency in a selected sample of drug naïve schizophrenia and comparing them with a healthy control group. We also aimed at assessing the influence of age, gender, family history of schizophrenia and duration of untreated illness on these parameters.
2. Materials and methods The study was carried out at a teaching cum tertiary care hospital in South India between JuneeDecember 2013. The hospital which is attached to a centrally funded autonomous university has all the specialty departments functioning within a single campus. Most of the service users are patients who can directly walk in without a prior appointment, make a case file and seek consultation for their health issues. This was a collaborative interdepartmental project between the departments of Anatomy and Psychiatry of the institute. Prior approval for the work was obtained from the Institute Human Ethics Committee. All patients attending the Psychiatry outpatient clinic first undergo a screening interview by a senior resident psychiatrist (equivalent to registrar) for psychiatric morbidity. Those who require detailed evaluation for making a diagnosis are allotted an appointment at a separate date when they would be asked to come with a reliable informant for diagnostic work up. On this day, a junior resident (equivalent to post graduate registrar or trainee) would first evaluate the case following which the case would be discussed with a consultant psychiatrist to formulate a psychiatric diagnosis and prepare a management plan. Hence, all the patients go through a two-stage assessment process involving clinical interviews before arriving at a diagnosis. Clinical diagnoses are made as per International Classification of Diseases (ICD) e 10 (WHO, 1992). For the present study, 40 consecutive patients who had thus received a diagnosis of first episode of schizophrenia, also confirmed as per the MiniInternational Neuropsychiatric Interview (M.I.N.I) (Sheehan et al., 1998), within the age group of 18e65 years were recruited. None of them had received prior treatment with any psychotropic agent such as anti-psychotics or even anti-depressants. This aspect was double checked by collecting information independently from the patient and a key informant. All patients who had taken any consultation with a psychiatrist or neurologist in the past were excluded in order to be sure about their drug naïve status. Informed consent was obtained in the vernacular from patient or the caregiver as appropriate. They constituted the cases for the present study. An equal number of age and gender matched apparently healthy individuals, selected from consenting hospital staff, were the controls for the study. All of them screened negative for psychiatric morbidity as per the M.I.N.I-Screen (Sheehan et al., 1998) (all interviews conducted by VM) as well as medical morbidity by scrutiny of their physical or electronic health records which are available for every staff member working in the institute. Relevant socio-demographic and clinical data including the duration of illness for the cases was extracted using a semi-structured proforma. Family history of schizophrenia along a three generation pedigree chart was assessed by interviewing the informant and confirmed using the same ICD-10 criteria as used for the cases. Subsequently, 5 ml of blood was drawn from both the cases and controls for assessing DNA damage and repair efficiency as per comet assay procedure detailed below. 2.1. Alkaline comet assay The procedure of Single cell gel electrophoretic assay by (Nandhakumar et al., 2011) was used for this study with minor modifications. Principle: Treatment of agarose-embedded lymphocytes with a hypertonic lysis solution and a detergent removes their cell membranes, cytoplasm, nuclear membrane and nucleoplasm thus exposing the nucleoid. When this nucleoid is treated with a highly alkaline solution like Sodium Hydroxide (pH-10), the DNA supercoils unwind thus exposing the alkali labile sites (apurinic/apyrimidinic sites) which appear as breaks in the nucleic acid structure.
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Fig. 1. (A) - Parts of a typical comet. CL e Total comet length, HD e Head diameter of comet, TL e Tail length of comet. 1(B) - Notepad file generated by Cometscore software showing comet parameters of five comets.
Such fragments of DNA which are negatively charged migrate towards the anode (positive electrode) when exposed to a suitable current during electrophoresis thereby producing a ‘comet’-like appearance. Blood collection, Lymphocyte separation and Cell Lysis: 5 ml of blood was collected under strict aseptic conditions from a peripheral vein of the subject using a syringe flushed with heparin (0.1 ml/5 ml blood). Lymphocytes were separated by centrifuging at 1500 rpm for 30 min using lymphocyte separation media (Histopaque 1077, density e 1.077 g/ml, Sigma) and used without storage. 20 mL of buffy coat was directly sandwiched between agarose gel layers on each of the 3 slides per patient (Triplicate assay) for alkaline comet assay. To examine the DNA repair, the remaining cells were incubated for 2 h in a nutrient medium containing RPMI 1640 Medium with L-glutamine (Sigma), fetal bovine serum suitable for Cell culture F2442 (Sigma) Penicillin and Streptomycin (Sigma). The suspension was again centrifuged and the cell pellet was subjected to conventional comet assay (triplicate assay). Cell membrane and nuclear membrane was lysed using a solution containing 2.5 M Sodium Chloride, 100 mM disodium EDTA, 10 mM Tris, Sodium Hydroxide pellets and TRITON X-100. Single cell gel electrophoresis: Electrophoresis was carried out in a horizontal submarine gel electrophoresis system in ice cold (4 C) alkaline medium containing Sodium Hydroxide (10 N) and 200 mM disodium EDTA (pH-10) for 30 min with 0.74 V/cm (between electrodes) and 300 mA after alkaline unwinding for 30 min in the same solution. DNA damage assessment: The slides were washed three times for 5 min each, with 0.4 M TriseHCl (pH 7.5), fixed with a solution containing trichloroacetic acid (TCA), zinc sulphate and glycerol and stained with Silver nitrate. The stained slides were observed
Table 1 Distribution of baseline characteristics of the sample. Variable Age (years)
Body weight (kg)
Gender Family history of schizophrenia Nicotine dependence Alcohol dependence
<20 21e30 31e40 41e50 More than 50 <50 51e60 61e70 71e80 More than 80 Male Female Present Absent Yes No Yes No
Cases n (%)
Controls n (%)
3 (7.5%) 21 (52.5%) 11 (27.5%) 4 (10%) 1 (2.5%) 4 (10%) 7 (17.5%) 14 (35%) 8 (20%) 7 (17.5%) 21 (52.5%) 19 (47.5%) 9 (22.5%) 31 (77.5%) 8 (20%) 32 (80%) 11 (27.5%) 29 (72.5%)
3 (7.5%) 21 (52.5%) 11 (27.5%) 4 (10%) 1 (2.5%) 6 (15%) 5 (12.5%) 13 (32.5%) 11 (27.5%) 5 (12.5%) 21 (52.5%) 19 (47.5%) e e e e e e
under a bright-field light microscope (Olympus BX43) and the images of 50 comets were captured per case before and after incubation randomly from the 3 slides using a CCD camera and analyzed using Cometscore Software e Version 1.5 (TriTek Corp., Stewart Road, Sumerduck, VA 22742). This software offers simple comet assay image analysis where the JPEG or TIFF images captured using the CCD camera are loaded into the software one at a time, and individual comets are selected manually with the a 2-click bounding rectangle scoring technique done on each comet at a time. Raw data which includes Total Comet length (mm), Head diameter of comet (mm), Tail length of comet (mm) and % of DNA in head and tail of comet are generated by the Cometscore software as a Notepad file (Fig. 1). The total length 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 towards the anode i.e. the damaged DNA. The data generated is automatically exported to a standard spreadsheet file. To minimize the subjective variations in the comet scoring method and measurement of DNA damage using the software, the procedure was done independently by two of the investigators (AM and PC) and the mean scores were used for analysis. Five comet parameters, namely the total comet length, head diameter, tail length, percentage (%) of DNA in head and tail of comet were calculated by two trained investigators (AM and PC) independently and their mean readings were used for the final analysis. The outcome assessors were unaware of the case or control status of the sample. Data was analyzed using the Statistical Package for Social Sciences (SPSS) - PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc. Normality of data was assessed using the ShapiroeWilk test. Continuous data was expressed as mean with standard deviation or median with interquartile range and compared between groups using the student t test or ManneWhitney U test depending on their distribution. Similarly, comparison of comet parameters before and after incubation was done using the paired t test or Wilcoxon signed rank test depending on data distribution. For the purpose of analysis, percentages of DNA in head and tail of comet was considered as continuous data and corrected till two decimal points similar to the other comet parameters. We also attempted to stratify the sample based on family history (presence or absence of schizophrenia) and duration of untreated illness (less than or more than 6 months) in order to discern the influence of these variables on comet parameters and DNA repair efficiency. All statistical analysis was done at 95% confidence interval and p < 0.05 was considered significant. 3. Results The mean age of the cases was 30.55 ± 8.47 years. The mean age of males and females was 28.90 ± 8.22 and 32.37 ± 8.58, respectively. 47% of the cases were females (n ¼ 19). The age and gender
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Fig. 2. Shows Silver stained comets in (A) Cases e Baseline DNA damage, (B) Cases e After DNA repair and (C) Controls e Baseline DNA damage.
distribution were similar in the control group (Table 1). The duration of illness ranged from a minimum of 1 month to a maximum of 72 months (median e 3 months, interquartile range e 11). The distribution of other baseline characteristics of the sample is shown in Table 1.
groups were not significantly different (for total Comet length U ¼ 118.00, p ¼ 0.05, for Head Diameter U ¼ 155.00, p ¼ 0.36, for Tail length U ¼ 132.00, p ¼ 0.12, for Percentage of DNA in head t ¼ 0.01, p ¼ 0.99, for Percentage of DNA in tail t ¼ 0.02, p ¼ 0.99). 3.2. DNA repair capacity
3.1. Baseline comet parameters The captured silver stained images of comets from lymphocytes of patients before and after DNA repair and baseline DNA damage in controls (sample image shown in Fig. 2) were used for the study. The baseline comet parameters in cases and controls are shown in Table 2. Notably, all the parameters tested except head diameter were significantly different in the cases and pointed to greater baseline DNA damage in this group. No significant gender differences were observed in any of the comet parameters (for total Comet length U ¼ 137.00, p ¼ 0.09, for Head Diameter U ¼ 143.00, p ¼ 0.13, for Tail length U ¼ 153.00, p ¼ 0.21, for Percentage of DNA in head t ¼ 0.32, p ¼ 0.75, for Percentage of DNA in tail t ¼ 0.33, p ¼ 0.74). Age did not correlate significantly with any of the comet indices (for total Comet length rs ¼ 0.08, p ¼ 0.62, for Head Diameter rs ¼ 0.24, p ¼ 0.14, for Tail length rs ¼ 0.06, p ¼ 0.69, for Percentage of DNA in head r ¼ 0.19, p ¼ 0.25, for Percentage of DNA in tail r ¼ 0.18, p ¼ 0.25). Next, stratified analysis was done based on presence (n ¼ 9) or absence (n ¼ 31) of family history of schizophrenia. We found that except for the total comet length and head diameter both of which were significantly lesser (U ¼ 34.00, p ¼ 0.001 and U ¼ 52.00, p ¼ 0.005 respectively) in patients with a positive family history of schizophrenia, the other comet parameters were comparable between the two groups (U ¼ 84.00, p ¼ 0.072 for tail length, t ¼ 1.295, 0.203 for percentage of DNA in head and t ¼ 1.282, p ¼ 0.207 for percentage of DNA in tail). Subsequently, stratification of the sample was done based on the duration of untreated illness. Two groups were obtained e with illness duration less than 6 months (n ¼ 25) or more than 6 months (n ¼ 15). The baseline comet parameters were compared between these groups. It was observed that the duration of illness did not exert any influence over the assayed parameters and the two
Table 2 Comparison of baseline comet parameters between patients & controls. Comet parameters
Group
Mean
SD
Comparison (p-value)
Total length of Comet (mm) Head diameter (mm)
Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases
76.35 126.84 73.06 76.70 91.38 76.33 6.40 50.35 8.62 23.69
17.43 39.71 11.29 21.37 4.66 10.76 9.16 32.38 4.66 10.81
U ¼ 114.50 (<0.001a)
% of DNA in Head Tail length (mm) % of DNA in Tail
U ¼ 695.50 (0.315) t ¼ 8.11 (<0.001a) U ¼ 41.50 (<0.001a) t ¼ 8.09 (<0.001a)
a Statistically significant; Comparisons done using Independent Student's t or Mann Whitney U Test.
Following incubation in the nutrient medium, all the comet parameters were found to significantly differ (Z ¼ 4.535, p < 0.001 for total comet length, Z ¼ 2.358, p ¼ 0.02 for head diameter, Z ¼ 3.098, p ¼ 0.01 for tail length, t ¼ 3.858, p < 0.001 for Percentage of DNA in head and t ¼ 3.861, p < 0.001 for Percentage of DNA in tail) from baseline among the cases (Fig. 3). Both males and females were comparable in their DNA repair capacity and did not differ significantly (for total Comet length U ¼ 164.00, p ¼ 0.34, for Head Diameter U ¼ 186.00, p ¼ 0.71, for Tail length U ¼ 155.00, p ¼ 0.23, for Percentage of DNA in head t ¼ 0.07, p ¼ 0.95, for Percentage of DNA in tail t ¼ 0.07, p ¼ 0.95). The DNA repair efficiency was also compared between groups stratified on the basis of a positive family history of schizophrenia and duration of untreated illness. A positive family history of schizophrenia (n ¼ 9) distinguished those with poor DNA repair capacity (for total comet length Z ¼ 0.840, p ¼ 0.401, for head diameter Z ¼ 0.280, p ¼ 0.779, for tail length Z ¼ 0.420, p ¼ 0.674, for Percentage of DNA in head t ¼ 1.800, p ¼ 0.110, for Percentage of DNA in tail t ¼ 1.800, p ¼ 0.110 among this group). In other words, those with a family history of schizophrenia demonstrated significantly decreased ability to repair DNA damage when incubated in the nutrient medium. The duration of illness exerted a marginal influence on the repair capacity (Table 3). Only 2 parameters (comet length and head diameter) were significantly different post incubation among those with a longer duration of illness (n ¼ 15) while all except head diameter were significantly repaired by patients presenting with a shorter duration of untreated illness (n ¼ 25). 4. Discussion Few studies have examined DNA damage and repair efficiency in drug naïve schizophrenia patients. Due to the practical difficulties involved in accessing brain cells, it has been suggested that peripheral lymphocytes can be used to reflect the overall condition of an organism as they constantly circulate and interact with various body tissues (Collins et al., 2008). Specifically, Yoon et al. (2010) have suggested that measurement of oxidative damage in the DNA of circulating lymphocytes can represent overall oxidative stress in the body. Hence, we have chosen to look at DNA damage and repair efficiency in the circulating peripheral lymphocytes of patients with schizophrenia due to their easier access. The mean age of males, in our sample, was slightly less than females. This is consistent with the reports from classic papers that males tend to have a younger mean age of onset of schizophrenia €fner et al., 1993). No specific gender compared to females (Ha predilection for the disorder is known and this is also reflected in
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Fig. 3. DNA repair capacity in drug naïve patients with schizophrenia.
the comparable gender distribution of the study sample (Abel et al., 2010). All the baseline comet parameters were significantly higher among patients than controls. Previous reviews have alluded to this finding by documenting increased oxidative damage in early schizophrenia (Boskovic et al., 2011; Ciobica et al., 2011). However, Psimadas and colleagues, in their study on 20 medicated chronic schizophrenics and matched controls, reported no baseline differences in DNA damage but their sample composed of individuals who were continuously ill and on psychotropics for at least 5 years (Psimadas et al., 2004). We did not notice any gender differences at baseline among patients in the extent of observed DNA damage. This resonates with the findings of a meta-analysis by Laurie and Abukmeil who conclude that the status of gender as a potential confounder is unclear (Lawrie and Abukmeil, 1998). Those with a heritable predisposition to schizophrenia showed greater baseline DNA damage. No significant differences in comet parameters were noted between groups in a stratified analysis based on duration of illness. No correlation was observed between age and parameters representing DNA damage. Our results show that significant baseline DNA damage already exists in drug naive schizophrenics and its extent is not significantly influenced by either age or gender. It was observed that the DNA repair efficiency was preserved in the lymphocytes of patients and all the comet parameters showed a significant change following incubation in the nutrient
medium. These findings are congruent with a previous report about DNA repair rates being comparable in dividing lymphoblasts of schizophrenia patients and healthy controls but the authors studied only markers of DNA-DSB's (Catts et al., 2012). Furthermore, our data show that those with a positive family history and longer duration of illness demonstrated significantly decreased capacity to repair the accumulated DNA damage. Due to the smaller numbers in the subgroups, our findings should be viewed as preliminary and require further replication by researchers in larger samples. Nevertheless, as pointed out by Sun et al. (2014), a pre-specified subgroup analysis carries greater credibility than an exploratory analysis and therefore these results may be given due weightage. A longer duration of untreated psychosis and positive family history of schizophrenia are well documented poor prognostic markers in schizophrenia (Stephens, 1978; Singh, 2007). However, poor DNA repair efficiency in schizophrenia has not been linked to these variables so far. The few previous studies that have investigated this issue have used different techniques to study DNA repair efficiency and thrown up conflicting results. While Zasukhina (1987) and Topinka et al. (1991) have shown significant defects in repair efficiency in schizophrenic patients in comparison to controls, Magin et al. (1991) failed to demonstrate a difference between the two groups. Only one previous study has evaluated DNA repair efficiency in schizophrenia using a similar methodology and invitro stimulating agent like ours. Their sample size
Table 3 Comparison of DNA repair efficiency in patients stratified according to duration of illness. Comet parameters Comet length Head diameter Percentage of DNA In head Tail length Percentage of DNA In tail a
Duration more than 6 months (n ¼ 15) Before After Before After Before After Before After Before After
123.27 102.84 71.99 63.73 76.06 77.74 51.33 39.34 23.94 22.25
± ± ± ± ± ± ± ± ± ±
56.37 22.87 16.85 10.00 10.21 8.52 47.05 23.34 10.21 8.52
Comparison (p-value)
Duration less than 6 months (n ¼ 25)
Z ¼ 2.271a (0.023a)
128.78 114.07 79.23 76.39 76.48 81.43 49.83 38.07 23.55 18.56
Z ¼ 2.062a (0.039a) t ¼ 1.349 (0.201) Z ¼ 1.153 (0.249) t ¼ 1.349 (0.200)
± ± ± ± ± ± ± ± ± ±
28.22 23.18 23.36 18.49 11.24 9.20 21.99 17.75 11.31 9.20
Statistically significant; all values expressed as mean ± SD; Comparisons done using Paired Student's t or Wilcoxon signed-rank test (Z).
Comparison (p-value) Z ¼ 3.873 (<0.001a) Z ¼ 1.384 (0.166) t ¼ 3.741 (0.001a) Z ¼ 2.832a (0.005a) t ¼ 3.744a (0.001a)
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was smaller and the results did not show any significant differences between patients and healthy controls (Psimadas et al., 2004). The findings of the present research must be viewed in the backdrop of its limitations. Firstly, convenient sampling was used and the sample size was limited. This may have contributed to the lack of significant findings in some of the subgroup analysis. No formal measure of the severity of schizophrenia was used and therefore its correlation with the extent of DNA damage could not be assessed. Subjective variations in the comet scoring method and measurement of DNA damage using the software are unavoidable. However, we sought to minimize this variability by having two of the investigators (AM and PC) independently score the parameters and then using their mean scores. Due to resource constraints, we were unable to report the DNA damage as lesions per 106 (one million) base pairs which would have allowed readers to decipher the levels of baseline genomic damage to the cells. The strengths of the study include the sampling of drug naïve schizophrenia patients in order to eliminate potential confounders with regard to genomic integrity. We also included an age and gender matched comparison group for methodological rigor. Certainly, ours is one of the very few studies that have also assessed DNA repair efficiency in schizophrenia. 5. Conclusion The present study shows that drug naïve patients with schizophrenia have significantly greater baseline DNA damage when compared to matched controls. Parameters such as age, gender and duration of illness do not seem to affect the degree of DNA damage at clinical presentation while those with a family history of schizophrenia have some indicators pointing towards increased DNA damage. We also found evidence that the DNA repair machinery is preserved in most patients and did not differ between the genders. Positive family history of schizophrenia had a stronger influence on genomic repair efficiency when compared to longer duration of untreated illness. Future work should focus upon the prospective impact of antipsychotics on markers of neurodegenerative brain changes. Additionally, studying the effect of adjunctive neuroprotective agents such as fatty acids and anti-oxidants in schizophrenia may aid our understanding and possibly help in identifying new targets for treatment and prevention of schizophrenia. Role of funding source The study was funded by an intramural grant from the institute (Sanction circular No.Edn.7 (1)/2013 dated 05.02.2013) that went towards the purchase of test equipments and consumables. Contributors AM conceptualized the idea, performed the comet assay technique, collected the relevant data and wrote the first draft of the manuscript. VM extracted articles from peer reviewed literature, helped in streamlining the case flow, performed data analysis and contributed to the intellectual content of the manuscript. RPR identified, referred the cases and revised the manuscript for important content. PC performed the comet assay technique independently to reduce bias and contributed to the final draft of the manuscript. Conflict of interest The authors report no conflict of interest.
Acknowledgments The study was funded by an intramural grant from the institute (Sanction circular No.Edn.7 (1)/2013 dated 05.02.2013). We thank the institute authorities for providing the necessary resources for the conduct of this research. References Abel KM, Drake R, Goldstein JM. Sex differences in schizophrenia. Int Rev Psychiatry Abingdon Engl 2010;22:417e28. Boskovic M, Vovk T, Kores Plesnicar B, Grabnar I. Oxidative stress in schizophrenia. Curr Neuropharmacol 2011;9:301e12. Buckley PF. Neuroimaging of schizophrenia: structural abnormalities and pathophysiological implications. Neuropsychiatr Dis Treat 2005;1:193e204. Catts VS, Catts SV, Jablensky A, Chandler D, Weickert CS, Lavin MF. Evidence of aberrant DNA damage response signalling but normal rates of DNA repair in dividing lymphoblasts from patients with schizophrenia. World J Biol Psychiatry 2012;13:114e25. Christopoulos I, Massouri G, Fotopoulos V, Hamogeorgakis T. A neurodegenerative perspective on schizophrenia. Ann Gen Psychiatry 2006;5:S261. Ciobica A, Padurariu M, Dobrin I, Stefanescu C, Dobrin R. Oxidative stress in schizophrenia - focusing on the main markers. Psychiatr Danub 2011;23: 237e45. F, Mancuso M, Siciliano G, Migliore L, Murri L. Genes and the environment Coppede in neurodegeneration. Biosci Rep 2006;26:341e67. Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, et al. The comet assay: topical issues. Mutagenesis 2008;23:143e51. Dadheech G, Mishra S, Gautam S, Sharma P. Evaluation of antioxidant deficit in schizophrenia. Indian J Psychiatry 2008;50:16e20. Fatemi SH, Folsom TD. The neurodevelopmental hypothesis of schizophrenia, Revisited. Schizophr Bull 2009;35:528e48. Fendri C, Mechri A, Khiari G, Othman A, Kerkeni A, Gaha L. Oxidative stress phale 2006;32: involvement in schizophrenia pathophysiology: a review. L'Ence 244e52. €fner H, Maurer K, Lo € ffler W, Riecher-Ro € ssler A. The influence of age and sex on Ha the onset and early course of schizophrenia. Br J Psychiatry J Ment Sci 1993;162: 80e6. Harrison PJ. The neuropathology of schizophrenia. A critical review of the data and their interpretation, Brain 1999;122:593e624. Jorgensen A, Broedbaek K, Fink-Jensen A, Knorr U, Soendergaard MG, Henriksen T, et al. Increased systemic oxidatively generated DNA and RNA damage in schizophrenia. Psychiatry Res 2013;209:417e23. Kasai K, Shenton ME, Salisbury DF, Hirayasu Y, Lee C-U, Ciszewski AA, et al. Progressive decrease of left superior temporal gyrus gray matter volume in patients with first-episode schizophrenia. Am J Psychiatry 2003;160: 156e64. Kidson C, Chen P. DNA damage, DNA repair and the genetic basis of Alzheimer's disease. Prog Brain Res 1986;70:291e301. Lawrie SM, Abukmeil SS. Brain abnormality in schizophrenia. A systematic and quantitative review of volumetric magnetic resonance imaging studies. Br J Psychiatry J Ment Sci 1998;172:110e20. Lindahl T. Instability and decay of the primary structure of DNA. Nature 1993;362: 709e15. Liu Z, Martin LJ. Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy. J Comp Neurol 2001;432:35e60. Madabhushi R, Pan L, Tsai L. DNA damage and its links to neurodegeneration. Neuron 2014;83:266e82. Magin GK, Robison SH, Breslin N, Wyatt RJ, Alexander RC. DNA repair and mutant frequency in schizophrenia. Mutat Res 1991;255:241e6. Martin LJ, Pan Y, Price AC, Sterling W, Copeland NG, Jenkins NA, et al. Parkinson's disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death. J Neurosci 2006;26:41e50. Martin LJ, Liu Z, Chen K, Price AC, Pan Y, Swaby JA, et al. Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death. J Comp Neurol 2007;500:20e46. Martin LJ. DNA damage and repair: relevance to mechanisms of neurodegeneration. J Neuropathol Exp Neurol 2008;67:377e87. Martin LJ, Liu Z, Pipino J, Chestnut B, Landek MA. Molecular regulation of DNA damage-induced apoptosis in neurons of cerebral cortex. Cereb Cortex 2009;19: 1273e93. Nandhakumar S, Parasuraman S, Shanmugam MM, Rao KR, Chand P, Bhat BV. Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). J Pharmacol Pharmacother 2011;2:107e11. Nishioka N, Arnold SE. Evidence for oxidative DNA damage in the hippocampus of elderly patients with chronic schizophrenia. Am J Geriatr Psychiatry 2004;12: 167e75. Picada JN, Dos Santos B de JN, Celso F, Monteiro JD, Da Rosa KM, Camacho LR, et al. Neurobehavioral and genotoxic parameters of antipsychotic agent aripiprazole in mice. Acta Pharmacol Sin 2011;32:1225e32.
A. Muraleedharan et al. / Journal of Psychiatric Research 68 (2015) 47e53 €w B, Karlsson HL, Mo € ller L, et al. The Polischouk AG, Holgersson A, Zong D, Stenerlo antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non small cell lung carcinoma cells to DNA double-strand break induced cell death. Mol Cancer Ther 2007;6:2303e9. Psimadas D, Messini-Nikolaki N, Zafiropoulou M, Fortos A, Tsilimigaki S, Piperakis SM. DNA damage and repair efficiency in lymphocytes from schizophrenic patients. Cancer Lett 2004;204:33e40. Rao KS. Genomic damage and its repair in young and aging brain. Mol Neurobiol 1993;7:23e48. Sahin E, DePinho RA. Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature 2010;464:520e8. Schnieder TP, Dwork AJ. Searching for neuropathology: gliosis in schizophrenia. Biol Psychiatry 2011;69:134e9. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E. The MiniInternational Neuropsychiatric Interview (M.I.N.I): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry 1998;59:22e33. Shenton ME, Dickey CC, Frumin M, McCarley RW. A review of MRI findings in schizophrenia. Schizophr Res 2001;49:1e52. Singh SP. Outcome measures in early psychosis: relevance of duration of untreated psychosis. Br J Psychiatry 2007;191:s58e63. Stephens JH. Long-term prognosis and followup in schizophrenia. Schizophr Bull 1978;4:25e47.
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Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay Aparna Muraleedharan a, Vikas Menon b, *, Ravi Philip Rajkumar b, Parkash Chand a a b
Department of Anatomy, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Dhanvantary Nagar, Puducherry, 605006, India Department of Psychiatry, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Dhanvantary Nagar, Puducherry, 605006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2015 Received in revised form 10 May 2015 Accepted 12 May 2015
The etiology of schizophrenia continues to be confounding and elusive. Some knowledge gaps exist in the neurodegenerative theory of schizophrenia. Oxidative DNA damage and repair deficits are relevant to the mechanisms of neurodegeneration but have not been studied in drug naïve schizophrenia. The present study used the comet assay technique to study the extent of DNA damage in circulating peripheral lymphocytes of patients with drug naïve schizophrenia (n ¼ 40) along with an age and gender matched control group (n ¼ 40). We also assessed the DNA repair efficiency in cases following incubation in a nutrient medium. All the assayed comet parameters demonstrated significantly greater baseline DNA damage in cases in comparison to the controls except for head diameter (p < 0.001 for all significant results, p ¼ 0.32 for head diameter). Gender, age and duration of illness (p ¼ 0.21, 0.69 and 0.12 respectively for tail length) did not influence any of the parameters significantly. Significant decrease was noted in the comet tail length and percentage of DNA in comet tail (p < 0.001 for both) in cases following incubation suggesting that the DNA repair machinery was preserved. No difference in DNA repair efficiency was noted between the genders (p ¼ 0.23 for tail length). Our findings confirm the presence of significant baseline DNA damage in schizophrenia even prior to the initiation of anti-psychotic treatment. Additionally, intact genomic repair efficiency was noted in this group as a whole. These results provide some evidence for oxidative DNA damage as molecular link underpinning neurodegeneration in drug naïve schizophrenia. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Schizophrenia Drug naive DNA damage Genetic repair Neurodegeneration
1. Introduction Schizophrenia is a chronic, debilitating psychiatric disorder whose etiology has been challenging to decipher. There are, broadly speaking, three hypothesis put forth to explain the etiopathogenesis of this condition. They are the neurodevelopmental (Fatemi and Folsom, 2009), neurodegenerative (Christopoulos et al., 2006) and the progressive neurodevelopmental model (Woods, 1998). The term neurodegeneration refers to chronic, progressive disorders of the nervous system characterized by continuing neuronal loss. The prototype examples of such disorders are Alzheimer's et al., 2006). Numerous disease and Huntington's disease (Coppede
* Corresponding author. Dept. of Psychiatry, JIPMER, Dhanvantary Nagar, Puducherry 605006, India. þ91 9894410296 (mobile); fax: þ91 413 2272067. E-mail address: [email protected] (V. Menon). http://dx.doi.org/10.1016/j.jpsychires.2015.05.018 0022-3956/© 2015 Elsevier Ltd. All rights reserved.
lines of evidence support the neurodegenerative theory of schizophrenia. Longitudinal neuroimaging studies have been able to demonstrate progressive changes in the size and function of frontal and temporal lobes following illness onset lending credence to this theory (Shenton et al., 2001; Kasai et al., 2003; Buckley, 2005). Oxidative stress is a hallmark of any neurodegenerative process and is also believed to be involved in the pathophysiology of schizophrenia. Evidence has accumulated regarding the decreased levels of endogenous anti-oxidants such as superoxide dismutase, glutathione and catalase in people with schizophrenia (Fendri et al., 2006; Dadheech et al., 2008; Tsai et al., 2013). On a conflicting note, gliosis which is another feature of neurodegeneration has been notably absent in histopathological models of schizophrenia indicative of knowledge gaps about this hypothesis (Harrison 1999; Schnieder and Dwork, 2011). Deoxyribonucleic Acid (DNA) damage can be defined as any alteration in DNA that induces a change in its function or modifies
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its coding properties (Lindahl, 1993; Rao, 1993). Compelling evidence, from animal and invitro models, suggest that DNA strand breaks are early antecedents of both apoptoptic and necrotic forms of neuronal cell death (Liu and Martin, 2001; Martin et al., 2006). Studies using cultured embryonic mouse cortical neurons have demonstrated the rapid accumulation of increasing DNA-single strand breaks (DNA-SSBs) with increasing levels of oxidative stress prior to eventual cell death. Furthermore, the timing of these genomic strand breaks in neurodegenerative disease models suggests that they are forerunners of a p53-driven apoptotic cascade in the cerebral cortex (Martin et al., 2009). Interestingly, authors who investigated mechanisms of motor neuron (MN) degeneration using transgenic mice expressing human mutant superoxide dismutase-1 (mSOD1) identified novel patterns of MN degeneration that differed from apoptosis structurally and biochemically. They concluded that there is a role for oxidative stress in the process of motor neuron degeneration induced by mSOD1 (Martin et al., 2007). Based on many of these findings, researchers have posited that oxidative stress induced genomic damage is relevant to the mechanisms resulting in neurodegeneration (Martin, 2008). Oxidative stress is thought to activate DNA damage pathways and play a critical role in activating telomere erosion, a key molecular correlate of the aging process (Sahin and DePinho, 2010). Some authors have suggested that this chronic state of oxidative stress on nucleic acids may underpin some of the clinical indicators of early aging in schizophrenia such as mildly accelerated cognitive decline (Jorgensen et al., 2013). DNA repair mechanisms have also been noted to be defective in many neurodegenerative conditions (Kidson and Chen, 1986). In a recent review that specifically focused on consequences of defective DNA repair mechanisms in non-proliferating cells such as neurons, the authors concluded that inefficient DNA repair has a robust link with aging and manifestations of classic neurodegenerative conditions such as Alzheimer's disease (Madabhushi et al., 2014). The evidence for neurodegeneration in schizophrenia remains sketchy. Previous investigators who studied urinary markers of oxidative DNA damage using ultra performance liquid chromatography with tandem mass spectroscopy concluded that there was a statistically significant increase in the excretion of these markers in schizophrenia patients when compared to matched controls (Jorgensen et al., 2013). Evidence for increased levels of oxidative DNA damage markers in schizophrenia have also come from studies on post mortem hippocampi in elderly patients with poor clinical outcomes (Nishioka and Arnold, 2004). By contrast, DNA repair response in schizophrenia has received comparatively less research attention. In a study using immortalized lymphoblasts from schizophrenia patients and healthy controls, the authors investigated the issue of DNA repair by studying temporal change in markers of DNA-DSBs following irradiation and concluded that there was no difference in rates of DNA repair between the two groups (Catts et al., 2012). Single cell gel electrophoresis or “comet” assay is regarded as a sensitive method for estimating the major types of DNA damage lesions in eukaryotic cells (Tice et al., 2000). Few studies have used this technique to measure levels of DNA damage and repair in schizophrenia. Further, most of the available data are from chronic schizophrenia patients who carry a lot of potential confounders that may contaminate these observations, particularly, antipsychotic treatment which has been shown to negatively influence genotoxic parameters in pre-clinical and clinical research (Polischouk et al., 2007; Picada et al., 2011). Hence, we undertook the present study with the twin objectives of assessing DNA damage and repair efficiency in a selected sample of drug naïve schizophrenia and comparing them with a healthy control group. We also aimed at assessing the influence of age, gender, family history of schizophrenia and duration of untreated illness on these parameters.
2. Materials and methods The study was carried out at a teaching cum tertiary care hospital in South India between JuneeDecember 2013. The hospital which is attached to a centrally funded autonomous university has all the specialty departments functioning within a single campus. Most of the service users are patients who can directly walk in without a prior appointment, make a case file and seek consultation for their health issues. This was a collaborative interdepartmental project between the departments of Anatomy and Psychiatry of the institute. Prior approval for the work was obtained from the Institute Human Ethics Committee. All patients attending the Psychiatry outpatient clinic first undergo a screening interview by a senior resident psychiatrist (equivalent to registrar) for psychiatric morbidity. Those who require detailed evaluation for making a diagnosis are allotted an appointment at a separate date when they would be asked to come with a reliable informant for diagnostic work up. On this day, a junior resident (equivalent to post graduate registrar or trainee) would first evaluate the case following which the case would be discussed with a consultant psychiatrist to formulate a psychiatric diagnosis and prepare a management plan. Hence, all the patients go through a two-stage assessment process involving clinical interviews before arriving at a diagnosis. Clinical diagnoses are made as per International Classification of Diseases (ICD) e 10 (WHO, 1992). For the present study, 40 consecutive patients who had thus received a diagnosis of first episode of schizophrenia, also confirmed as per the MiniInternational Neuropsychiatric Interview (M.I.N.I) (Sheehan et al., 1998), within the age group of 18e65 years were recruited. None of them had received prior treatment with any psychotropic agent such as anti-psychotics or even anti-depressants. This aspect was double checked by collecting information independently from the patient and a key informant. All patients who had taken any consultation with a psychiatrist or neurologist in the past were excluded in order to be sure about their drug naïve status. Informed consent was obtained in the vernacular from patient or the caregiver as appropriate. They constituted the cases for the present study. An equal number of age and gender matched apparently healthy individuals, selected from consenting hospital staff, were the controls for the study. All of them screened negative for psychiatric morbidity as per the M.I.N.I-Screen (Sheehan et al., 1998) (all interviews conducted by VM) as well as medical morbidity by scrutiny of their physical or electronic health records which are available for every staff member working in the institute. Relevant socio-demographic and clinical data including the duration of illness for the cases was extracted using a semi-structured proforma. Family history of schizophrenia along a three generation pedigree chart was assessed by interviewing the informant and confirmed using the same ICD-10 criteria as used for the cases. Subsequently, 5 ml of blood was drawn from both the cases and controls for assessing DNA damage and repair efficiency as per comet assay procedure detailed below. 2.1. Alkaline comet assay The procedure of Single cell gel electrophoretic assay by (Nandhakumar et al., 2011) was used for this study with minor modifications. Principle: Treatment of agarose-embedded lymphocytes with a hypertonic lysis solution and a detergent removes their cell membranes, cytoplasm, nuclear membrane and nucleoplasm thus exposing the nucleoid. When this nucleoid is treated with a highly alkaline solution like Sodium Hydroxide (pH-10), the DNA supercoils unwind thus exposing the alkali labile sites (apurinic/apyrimidinic sites) which appear as breaks in the nucleic acid structure.
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Fig. 1. (A) - Parts of a typical comet. CL e Total comet length, HD e Head diameter of comet, TL e Tail length of comet. 1(B) - Notepad file generated by Cometscore software showing comet parameters of five comets.
Such fragments of DNA which are negatively charged migrate towards the anode (positive electrode) when exposed to a suitable current during electrophoresis thereby producing a ‘comet’-like appearance. Blood collection, Lymphocyte separation and Cell Lysis: 5 ml of blood was collected under strict aseptic conditions from a peripheral vein of the subject using a syringe flushed with heparin (0.1 ml/5 ml blood). Lymphocytes were separated by centrifuging at 1500 rpm for 30 min using lymphocyte separation media (Histopaque 1077, density e 1.077 g/ml, Sigma) and used without storage. 20 mL of buffy coat was directly sandwiched between agarose gel layers on each of the 3 slides per patient (Triplicate assay) for alkaline comet assay. To examine the DNA repair, the remaining cells were incubated for 2 h in a nutrient medium containing RPMI 1640 Medium with L-glutamine (Sigma), fetal bovine serum suitable for Cell culture F2442 (Sigma) Penicillin and Streptomycin (Sigma). The suspension was again centrifuged and the cell pellet was subjected to conventional comet assay (triplicate assay). Cell membrane and nuclear membrane was lysed using a solution containing 2.5 M Sodium Chloride, 100 mM disodium EDTA, 10 mM Tris, Sodium Hydroxide pellets and TRITON X-100. Single cell gel electrophoresis: Electrophoresis was carried out in a horizontal submarine gel electrophoresis system in ice cold (4 C) alkaline medium containing Sodium Hydroxide (10 N) and 200 mM disodium EDTA (pH-10) for 30 min with 0.74 V/cm (between electrodes) and 300 mA after alkaline unwinding for 30 min in the same solution. DNA damage assessment: The slides were washed three times for 5 min each, with 0.4 M TriseHCl (pH 7.5), fixed with a solution containing trichloroacetic acid (TCA), zinc sulphate and glycerol and stained with Silver nitrate. The stained slides were observed
Table 1 Distribution of baseline characteristics of the sample. Variable Age (years)
Body weight (kg)
Gender Family history of schizophrenia Nicotine dependence Alcohol dependence
<20 21e30 31e40 41e50 More than 50 <50 51e60 61e70 71e80 More than 80 Male Female Present Absent Yes No Yes No
Cases n (%)
Controls n (%)
3 (7.5%) 21 (52.5%) 11 (27.5%) 4 (10%) 1 (2.5%) 4 (10%) 7 (17.5%) 14 (35%) 8 (20%) 7 (17.5%) 21 (52.5%) 19 (47.5%) 9 (22.5%) 31 (77.5%) 8 (20%) 32 (80%) 11 (27.5%) 29 (72.5%)
3 (7.5%) 21 (52.5%) 11 (27.5%) 4 (10%) 1 (2.5%) 6 (15%) 5 (12.5%) 13 (32.5%) 11 (27.5%) 5 (12.5%) 21 (52.5%) 19 (47.5%) e e e e e e
under a bright-field light microscope (Olympus BX43) and the images of 50 comets were captured per case before and after incubation randomly from the 3 slides using a CCD camera and analyzed using Cometscore Software e Version 1.5 (TriTek Corp., Stewart Road, Sumerduck, VA 22742). This software offers simple comet assay image analysis where the JPEG or TIFF images captured using the CCD camera are loaded into the software one at a time, and individual comets are selected manually with the a 2-click bounding rectangle scoring technique done on each comet at a time. Raw data which includes Total Comet length (mm), Head diameter of comet (mm), Tail length of comet (mm) and % of DNA in head and tail of comet are generated by the Cometscore software as a Notepad file (Fig. 1). The total length 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 towards the anode i.e. the damaged DNA. The data generated is automatically exported to a standard spreadsheet file. To minimize the subjective variations in the comet scoring method and measurement of DNA damage using the software, the procedure was done independently by two of the investigators (AM and PC) and the mean scores were used for analysis. Five comet parameters, namely the total comet length, head diameter, tail length, percentage (%) of DNA in head and tail of comet were calculated by two trained investigators (AM and PC) independently and their mean readings were used for the final analysis. The outcome assessors were unaware of the case or control status of the sample. Data was analyzed using the Statistical Package for Social Sciences (SPSS) - PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc. Normality of data was assessed using the ShapiroeWilk test. Continuous data was expressed as mean with standard deviation or median with interquartile range and compared between groups using the student t test or ManneWhitney U test depending on their distribution. Similarly, comparison of comet parameters before and after incubation was done using the paired t test or Wilcoxon signed rank test depending on data distribution. For the purpose of analysis, percentages of DNA in head and tail of comet was considered as continuous data and corrected till two decimal points similar to the other comet parameters. We also attempted to stratify the sample based on family history (presence or absence of schizophrenia) and duration of untreated illness (less than or more than 6 months) in order to discern the influence of these variables on comet parameters and DNA repair efficiency. All statistical analysis was done at 95% confidence interval and p < 0.05 was considered significant. 3. Results The mean age of the cases was 30.55 ± 8.47 years. The mean age of males and females was 28.90 ± 8.22 and 32.37 ± 8.58, respectively. 47% of the cases were females (n ¼ 19). The age and gender
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Fig. 2. Shows Silver stained comets in (A) Cases e Baseline DNA damage, (B) Cases e After DNA repair and (C) Controls e Baseline DNA damage.
distribution were similar in the control group (Table 1). The duration of illness ranged from a minimum of 1 month to a maximum of 72 months (median e 3 months, interquartile range e 11). The distribution of other baseline characteristics of the sample is shown in Table 1.
groups were not significantly different (for total Comet length U ¼ 118.00, p ¼ 0.05, for Head Diameter U ¼ 155.00, p ¼ 0.36, for Tail length U ¼ 132.00, p ¼ 0.12, for Percentage of DNA in head t ¼ 0.01, p ¼ 0.99, for Percentage of DNA in tail t ¼ 0.02, p ¼ 0.99). 3.2. DNA repair capacity
3.1. Baseline comet parameters The captured silver stained images of comets from lymphocytes of patients before and after DNA repair and baseline DNA damage in controls (sample image shown in Fig. 2) were used for the study. The baseline comet parameters in cases and controls are shown in Table 2. Notably, all the parameters tested except head diameter were significantly different in the cases and pointed to greater baseline DNA damage in this group. No significant gender differences were observed in any of the comet parameters (for total Comet length U ¼ 137.00, p ¼ 0.09, for Head Diameter U ¼ 143.00, p ¼ 0.13, for Tail length U ¼ 153.00, p ¼ 0.21, for Percentage of DNA in head t ¼ 0.32, p ¼ 0.75, for Percentage of DNA in tail t ¼ 0.33, p ¼ 0.74). Age did not correlate significantly with any of the comet indices (for total Comet length rs ¼ 0.08, p ¼ 0.62, for Head Diameter rs ¼ 0.24, p ¼ 0.14, for Tail length rs ¼ 0.06, p ¼ 0.69, for Percentage of DNA in head r ¼ 0.19, p ¼ 0.25, for Percentage of DNA in tail r ¼ 0.18, p ¼ 0.25). Next, stratified analysis was done based on presence (n ¼ 9) or absence (n ¼ 31) of family history of schizophrenia. We found that except for the total comet length and head diameter both of which were significantly lesser (U ¼ 34.00, p ¼ 0.001 and U ¼ 52.00, p ¼ 0.005 respectively) in patients with a positive family history of schizophrenia, the other comet parameters were comparable between the two groups (U ¼ 84.00, p ¼ 0.072 for tail length, t ¼ 1.295, 0.203 for percentage of DNA in head and t ¼ 1.282, p ¼ 0.207 for percentage of DNA in tail). Subsequently, stratification of the sample was done based on the duration of untreated illness. Two groups were obtained e with illness duration less than 6 months (n ¼ 25) or more than 6 months (n ¼ 15). The baseline comet parameters were compared between these groups. It was observed that the duration of illness did not exert any influence over the assayed parameters and the two
Table 2 Comparison of baseline comet parameters between patients & controls. Comet parameters
Group
Mean
SD
Comparison (p-value)
Total length of Comet (mm) Head diameter (mm)
Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases
76.35 126.84 73.06 76.70 91.38 76.33 6.40 50.35 8.62 23.69
17.43 39.71 11.29 21.37 4.66 10.76 9.16 32.38 4.66 10.81
U ¼ 114.50 (<0.001a)
% of DNA in Head Tail length (mm) % of DNA in Tail
U ¼ 695.50 (0.315) t ¼ 8.11 (<0.001a) U ¼ 41.50 (<0.001a) t ¼ 8.09 (<0.001a)
a Statistically significant; Comparisons done using Independent Student's t or Mann Whitney U Test.
Following incubation in the nutrient medium, all the comet parameters were found to significantly differ (Z ¼ 4.535, p < 0.001 for total comet length, Z ¼ 2.358, p ¼ 0.02 for head diameter, Z ¼ 3.098, p ¼ 0.01 for tail length, t ¼ 3.858, p < 0.001 for Percentage of DNA in head and t ¼ 3.861, p < 0.001 for Percentage of DNA in tail) from baseline among the cases (Fig. 3). Both males and females were comparable in their DNA repair capacity and did not differ significantly (for total Comet length U ¼ 164.00, p ¼ 0.34, for Head Diameter U ¼ 186.00, p ¼ 0.71, for Tail length U ¼ 155.00, p ¼ 0.23, for Percentage of DNA in head t ¼ 0.07, p ¼ 0.95, for Percentage of DNA in tail t ¼ 0.07, p ¼ 0.95). The DNA repair efficiency was also compared between groups stratified on the basis of a positive family history of schizophrenia and duration of untreated illness. A positive family history of schizophrenia (n ¼ 9) distinguished those with poor DNA repair capacity (for total comet length Z ¼ 0.840, p ¼ 0.401, for head diameter Z ¼ 0.280, p ¼ 0.779, for tail length Z ¼ 0.420, p ¼ 0.674, for Percentage of DNA in head t ¼ 1.800, p ¼ 0.110, for Percentage of DNA in tail t ¼ 1.800, p ¼ 0.110 among this group). In other words, those with a family history of schizophrenia demonstrated significantly decreased ability to repair DNA damage when incubated in the nutrient medium. The duration of illness exerted a marginal influence on the repair capacity (Table 3). Only 2 parameters (comet length and head diameter) were significantly different post incubation among those with a longer duration of illness (n ¼ 15) while all except head diameter were significantly repaired by patients presenting with a shorter duration of untreated illness (n ¼ 25). 4. Discussion Few studies have examined DNA damage and repair efficiency in drug naïve schizophrenia patients. Due to the practical difficulties involved in accessing brain cells, it has been suggested that peripheral lymphocytes can be used to reflect the overall condition of an organism as they constantly circulate and interact with various body tissues (Collins et al., 2008). Specifically, Yoon et al. (2010) have suggested that measurement of oxidative damage in the DNA of circulating lymphocytes can represent overall oxidative stress in the body. Hence, we have chosen to look at DNA damage and repair efficiency in the circulating peripheral lymphocytes of patients with schizophrenia due to their easier access. The mean age of males, in our sample, was slightly less than females. This is consistent with the reports from classic papers that males tend to have a younger mean age of onset of schizophrenia €fner et al., 1993). No specific gender compared to females (Ha predilection for the disorder is known and this is also reflected in
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51
Fig. 3. DNA repair capacity in drug naïve patients with schizophrenia.
the comparable gender distribution of the study sample (Abel et al., 2010). All the baseline comet parameters were significantly higher among patients than controls. Previous reviews have alluded to this finding by documenting increased oxidative damage in early schizophrenia (Boskovic et al., 2011; Ciobica et al., 2011). However, Psimadas and colleagues, in their study on 20 medicated chronic schizophrenics and matched controls, reported no baseline differences in DNA damage but their sample composed of individuals who were continuously ill and on psychotropics for at least 5 years (Psimadas et al., 2004). We did not notice any gender differences at baseline among patients in the extent of observed DNA damage. This resonates with the findings of a meta-analysis by Laurie and Abukmeil who conclude that the status of gender as a potential confounder is unclear (Lawrie and Abukmeil, 1998). Those with a heritable predisposition to schizophrenia showed greater baseline DNA damage. No significant differences in comet parameters were noted between groups in a stratified analysis based on duration of illness. No correlation was observed between age and parameters representing DNA damage. Our results show that significant baseline DNA damage already exists in drug naive schizophrenics and its extent is not significantly influenced by either age or gender. It was observed that the DNA repair efficiency was preserved in the lymphocytes of patients and all the comet parameters showed a significant change following incubation in the nutrient
medium. These findings are congruent with a previous report about DNA repair rates being comparable in dividing lymphoblasts of schizophrenia patients and healthy controls but the authors studied only markers of DNA-DSB's (Catts et al., 2012). Furthermore, our data show that those with a positive family history and longer duration of illness demonstrated significantly decreased capacity to repair the accumulated DNA damage. Due to the smaller numbers in the subgroups, our findings should be viewed as preliminary and require further replication by researchers in larger samples. Nevertheless, as pointed out by Sun et al. (2014), a pre-specified subgroup analysis carries greater credibility than an exploratory analysis and therefore these results may be given due weightage. A longer duration of untreated psychosis and positive family history of schizophrenia are well documented poor prognostic markers in schizophrenia (Stephens, 1978; Singh, 2007). However, poor DNA repair efficiency in schizophrenia has not been linked to these variables so far. The few previous studies that have investigated this issue have used different techniques to study DNA repair efficiency and thrown up conflicting results. While Zasukhina (1987) and Topinka et al. (1991) have shown significant defects in repair efficiency in schizophrenic patients in comparison to controls, Magin et al. (1991) failed to demonstrate a difference between the two groups. Only one previous study has evaluated DNA repair efficiency in schizophrenia using a similar methodology and invitro stimulating agent like ours. Their sample size
Table 3 Comparison of DNA repair efficiency in patients stratified according to duration of illness. Comet parameters Comet length Head diameter Percentage of DNA In head Tail length Percentage of DNA In tail a
Duration more than 6 months (n ¼ 15) Before After Before After Before After Before After Before After
123.27 102.84 71.99 63.73 76.06 77.74 51.33 39.34 23.94 22.25
± ± ± ± ± ± ± ± ± ±
56.37 22.87 16.85 10.00 10.21 8.52 47.05 23.34 10.21 8.52
Comparison (p-value)
Duration less than 6 months (n ¼ 25)
Z ¼ 2.271a (0.023a)
128.78 114.07 79.23 76.39 76.48 81.43 49.83 38.07 23.55 18.56
Z ¼ 2.062a (0.039a) t ¼ 1.349 (0.201) Z ¼ 1.153 (0.249) t ¼ 1.349 (0.200)
± ± ± ± ± ± ± ± ± ±
28.22 23.18 23.36 18.49 11.24 9.20 21.99 17.75 11.31 9.20
Statistically significant; all values expressed as mean ± SD; Comparisons done using Paired Student's t or Wilcoxon signed-rank test (Z).
Comparison (p-value) Z ¼ 3.873 (<0.001a) Z ¼ 1.384 (0.166) t ¼ 3.741 (0.001a) Z ¼ 2.832a (0.005a) t ¼ 3.744a (0.001a)
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was smaller and the results did not show any significant differences between patients and healthy controls (Psimadas et al., 2004). The findings of the present research must be viewed in the backdrop of its limitations. Firstly, convenient sampling was used and the sample size was limited. This may have contributed to the lack of significant findings in some of the subgroup analysis. No formal measure of the severity of schizophrenia was used and therefore its correlation with the extent of DNA damage could not be assessed. Subjective variations in the comet scoring method and measurement of DNA damage using the software are unavoidable. However, we sought to minimize this variability by having two of the investigators (AM and PC) independently score the parameters and then using their mean scores. Due to resource constraints, we were unable to report the DNA damage as lesions per 106 (one million) base pairs which would have allowed readers to decipher the levels of baseline genomic damage to the cells. The strengths of the study include the sampling of drug naïve schizophrenia patients in order to eliminate potential confounders with regard to genomic integrity. We also included an age and gender matched comparison group for methodological rigor. Certainly, ours is one of the very few studies that have also assessed DNA repair efficiency in schizophrenia. 5. Conclusion The present study shows that drug naïve patients with schizophrenia have significantly greater baseline DNA damage when compared to matched controls. Parameters such as age, gender and duration of illness do not seem to affect the degree of DNA damage at clinical presentation while those with a family history of schizophrenia have some indicators pointing towards increased DNA damage. We also found evidence that the DNA repair machinery is preserved in most patients and did not differ between the genders. Positive family history of schizophrenia had a stronger influence on genomic repair efficiency when compared to longer duration of untreated illness. Future work should focus upon the prospective impact of antipsychotics on markers of neurodegenerative brain changes. Additionally, studying the effect of adjunctive neuroprotective agents such as fatty acids and anti-oxidants in schizophrenia may aid our understanding and possibly help in identifying new targets for treatment and prevention of schizophrenia. Role of funding source The study was funded by an intramural grant from the institute (Sanction circular No.Edn.7 (1)/2013 dated 05.02.2013) that went towards the purchase of test equipments and consumables. Contributors AM conceptualized the idea, performed the comet assay technique, collected the relevant data and wrote the first draft of the manuscript. VM extracted articles from peer reviewed literature, helped in streamlining the case flow, performed data analysis and contributed to the intellectual content of the manuscript. RPR identified, referred the cases and revised the manuscript for important content. PC performed the comet assay technique independently to reduce bias and contributed to the final draft of the manuscript. Conflict of interest The authors report no conflict of interest.
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