Effects of risperidone and haloperidol on superoxide dismutase and nitric oxide in schizophrenia

Neuropharmacology 62 (2012) 1928e1934

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Effects of risperidone and haloperidol on superoxide dismutase and nitric oxide in schizophrenia Xiang Yang Zhang a, b, c, d, *, Dong Feng Zhou a, **, Yu Cun Shen a, Pei Yan Zhang d, Wu Fang Zhang a, Jun Liang a, Da Chun Chen d, Mei Hong Xiu d, Therese A. Kosten b, Thomas R. Kosten b, *** a

Institute of Mental Health, Peking University, Beijing, PR China Menninger Department of Psychiatry and Behavioral Sciences, Baylor College of Medicine, Houston, Texas, USA Institute of Psychology, Chinese Academy of Sciences, Beijing, PR China d Beijing HuiLongGuan Hospital, Beijing, PR China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 October 2011 Received in revised form 5 December 2011 Accepted 10 December 2011

Oxidative stress may be involved in the pathophysiology of schizophrenia. No double-blind study has compared the effects of typical and atypical antipsychotics on both antioxidant enzyme activity and nitric oxide (NO) levels in schizophrenic patients. Seventy-eight inpatients with chronic schizophrenia were randomly assigned to 12 weeks of treatment with 6 mg/day of risperidone or 20 mg/day of haloperidol using a double-blind design. Clinical efficacy was determined using the Positive and Negative Syndrome Scale. Blood superoxide dismutase (SOD) and plasma NO levels were measured in patients and 30 normal controls. Our results showed that following a 2-week washout period, levels of SOD and NO were significantly increased in patients with schizophrenia compared to normal controls. Both risperidone and haloperidol equivalently reduced the elevated blood SOD levels in schizophrenia, but neither medication reduced the elevated plasma NO levels in schizophrenia. Low blood SOD levels at baseline predicted greater symptom improvement during treatment, and greater change in SOD was correlated with greater symptom improvement. These results suggest that both typical and atypical antipsychotic drugs may at least partially normalize abnormal free radical metabolism in schizophrenia, and some free radical parameters at baseline may predict antipsychotic responses of schizophrenic patients. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Schizophrenia Free radical Oxidative stress Antipsychotic treatment

1. Introduction The etiology of schizophrenia remains unclear, but oxidative stress due to an imbalance between radical-generating and radicalscavenging systems appears to be one contributor (Lohr, 1991; Reddy et al., 1991; Lohr and Browning, 1995; Mahadik and Mukherjee, 1996; Yao et al., 2003, 2004; Fendri et al., 2006; Ng et al., 2008; Bitanihirwe and Woo, 2011). Numerous studies show that patients with schizophrenia have altered antioxidant enzyme

* Corresponding author. VA Medical Center, Research Building 109, Room 130, 2002 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: þ1 7137911414x5824; fax: þ1 713 794 7938. ** Corresponding author. Institute of Mental Health, Peking University, 38 Huayuanbei Roa, HaiDian District, Beijing, PR China. Tel.: þ86 10 82801940; fax: þ86 10 62027314. *** Corresponding author. VA Medical Center, Research Building 110, Room 229, 2002 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: þ1 7137947032; fax: þ1 713 794 7938. E-mail addresses: [email protected] (X.Y. Zhang), [email protected] (D.F. Zhou), [email protected] (T.R. Kosten). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.12.014

activities and increased levels of lipid peroxidation. For example, superoxide dismutase (SOD) activity, a key enzyme involved in the detoxification of superoxide radicals, is elevated above normal levels in chronic schizophrenic patients (Reddy et al., 1992; Herken et al., 2001; Yao et al., 2001; Zhang et al., 2003aec, 2006; Gama et al., 2006; Kunz et al., 2008; Padurariu et al., 2010). Moreover, high levels of lipid peroxidation products are increased in plasma, serum (Mukherjee et al., 1996; Akyol et al., 2002; Gama et al., 2006; Ben Othmen et al., 2008; Dietrich-Muszalska and Kontek, 2010; Huang et al., 2010; Padurariu et al., 2010), red blood cells (Herken et al., 2001) and cerebrospinal fluid (CSF) of schizophrenic patients (Lohr, 1991). However, the results in the measure of free radical parameters are often contradictory. For example, decreased SOD activity was found in neuroleptic naive, first episode schizophrenic patients (Mukherjee et al., 1996; Reddy et al., 2003), in chronically-medicated patients (Akyol et al., 2002; Ranjekar et al., 2003; Ben Othmen et al., 2008; Zhang et al., 2006), or in chronically-unmedicated patients (Raffa et al., 2009). Several factors may be responsible for these discrepancies, such as differences in techniques of measuring oxidative parameters, differences in tested material (erythrocytes, vs. serum vs. plasma), sampling of

X.Y. Zhang et al. / Neuropharmacology 62 (2012) 1928e1934

patients in different stages of disease progression (acute vs. chronic or active phase vs. remission), exposure for neuroleptic treatment (naive vs. medicated vs. medication-free), different ethnic origin, lifestyle or dietary pattern (Reddy and Yao, 1996). Nitric oxide (NO) is biosynthesized endogenously from L-arginine and oxygen by various nitric oxide synthase (NOS) enzymes and by reduction of inorganic nitrate as a neurotransmitter, but it also can produce hydroxyl and nitrogen dioxide radicals (Yermolaieva et al., 2000; Lee and Kim, 2008). Its release produces vasodilation, inhibition of platelet aggregation, and both neuroprotection and neurotoxicity (Yermolaieva et al., 2000). Elevated NO production occurs in different neuropsychiatric disorders, such as Alzheimer’s disease, multiple sclerosis, Parkinson’s disease and some studies of schizophrenia (Akyol et al., 2004; Bernstein et al., 2005; Guix et al., 2005). However, conflicting findings have been reported for NO levels in schizophrenia. Most studies of blood samples from schizophrenic patients show increased NOS activity and the levels of NO metabolites, such as NOS activity in platelet (Das et al., 1995), plasma and serum nitrate and nitrite (Das et al., 1998; Zoroglu et al., 2002; Taneli et al., 2004), and NO in erythrocytes (Taneli et al., 2004). Moreover, lower blood concentrations of the NOS inhibitor, asymmetrical dimethylarginine (ADMA), have been reported (Das et al., 1998). Also, increased neuronal NO synthase (nNOS) activity (Das et al., 1995) and expression (Baba et al., 2004; Yao et al., 2004) as well as abnormal distribution of NOScontaining neurons (Bernstein et al., 2005) have been reported in brains of schizophrenia patients. However, a few studies have reported reduced NO metabolism in blood and diminished NO metabolite levels in the CSF of schizophrenia (Srivastava et al., 2001; Suzuki et al., 2003; Yanik et al., 2003; Ramirez et al., 2004; Lee and Kim, 2008). Hence, controversy persists on excess or deficient NO synthesis in schizophrenia (Bernstein et al., 2005). Antioxidant enzymes, such as SOD, catalase (CAT), and glutathione peroxidase (GSH-Px), lipid peroxidation levels, NO metabolites and NOS activities in the blood and brain can be substantively changed during short-term neuroleptic treatment in animal studies (Reddy et al., 1991; Bernstein et al., 2005). Animal studies have found that antipsychotic drugs increase free radical production through increased catecholamine turnover and medication metabolism (Lohr et al., 2003). These free radical increases are associated with increases in membrane lipid peroxidation products and brain levels of antioxidant enzymes (Cadet and Perumal, 1990). These medication effects may differ among the types of neuroleptics, since Parikh et al. (2003) showed increased lipid peroxidation and decreased SOD activity in the brain of rats chronically treated with haloperidol, but not with risperidone, olanzapine or clozapine. Similarly, Reinke et al. (2004) showed high thiobarbituric acid reactive substances (TBARS) levels in rat brain after haloperidol treatment, but not after clozapine. Furthermore, several, but not all, reports showed that haloperidol and chlorpromazine inhibit the neuronal NOS (nNOS) activity (Kuloglu et al., 2002; Lau et al., 2003; Nel and Harvey, 2003). Long-term (28 days) treatment with atypical antipsychotic drugs, including risperidone, olanzapine, and quetiapine do not influence rat brain levels of nNOS (Tarazi et al., 2002). Thus, free radical metabolism may differ between typical and atypical antipsychotics, although an important difference between these animals and schizophrenic patients is that the treated animals have normal antioxidant enzyme system. A few studies have examined neuroleptic effects on antioxidant enzymes and NO in schizophrenia, but have conflicting results. Using a within subject, repeated-measures design, Reddy and associates noted that neuroleptic withdrawal in schizophrenic patients lowered the initially high SOD activity toward normal levels (Reddy et al., 2003). However, Yao and associates using a similar design found that haloperidol treatment decreased SOD

1929

and GSH-Px activities (Yao et al., 1998), and our previous study found that initially high SOD activity decreased after 12 weeks of treatment with the atypical antipsychotic risperidone (Zhang et al., 2003b). On the other hand, more recently, Sarandol et al. (2007) reported that 6 weeks of treatment with either typical or atypical antipsychotics did not affect the elevated baseline SOD activity. Similarly, 6-weeks of treatment with atypical antipsychotics had no significant effects on serum NO metabolites in schizophrenia (Tarazi et al., 2002). A more recent study showed that 6 weeks of risperidone treatment increased the initially low plasma NO metabolite levels in schizophrenic patients, especially in those responders (Lee and Kim, 2008). Thus, antipsychotic agents do not have clear effects on the antioxidant enzymes or NO system in schizophrenia. We therefore have compared the effects of typical and atypical antipsychotic drugs on the antioxidant enzymes and NO system in drug-free schizophrenic patients before and after 12-weeks of treatment with fixed-dose risperidone and haloperidol in a double-blind, randomized clinical trial (Zhang et al., 2001). We also examined the relationships between psychopathologic symptom improvement and changes in SOD and NO levels, and whether SOD and NO levels at baseline could predict antipsychotic treatment responses. 2. Materials and methods 2.1. Subjects We enrolled 78 physically healthy Chinese in-patients who met the Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R) criteria for schizophrenia, as determined using the Structured Clinical Interview for DSM-III-R (SCID) from the Beijing Hui-Long-Guan Hospital, a Beijing City owned psychiatric hospital. All schizophrenic patients were chronic with an illness course of at least 5 years. Thirty healthy controls were recruited from the local community, and matched for age and gender. Current mental status and personal or family history of any mental disorder was assessed by a clinical psychiatrist. None of the healthy control subjects presented a personal or family history of psychiatric disorder. A complete medical history and physical examination, laboratory tests including a urine and blood screen were obtained from patients and control subjects. They were in good physical health, and any subjects with medical abnormalities were excluded. Neither the schizophrenic patients nor the control subjects suffered from substance abuse/dependence before they enrolled in the study. All subjects were Han Chinese being recruited at the same period from Beijing area. Their characteristics are summarized in Table 1. All subjects gave informed written consent to participate in the study, which was approved by the Institutional Review Board of the Institute of Mental Health, Peking University. 2.2. Clinical treatment and clinical ratings The study design has been described previously (Zhang et al., 2001). Patients had an equal probability of being assigned to the two groups. An independent third party placed them in either risperidone or haloperidol group according to a computer-generated randomization list compiled through simple randomization. Briefly the clinical trial had a 2-week placebo lead-in followed by random assignment to risperidone or haloperidol for 12 weeks of double-blind treatment. Eighty patients entered the study and 78 completed the 2-week placebo wash-in. The dose of risperidone (n ¼ 41) was increased to 6 mg/day and the dose of haloperidol (n ¼ 37) to 20 mg/day during the first week of blind administration, and the doses

Table 1 Demographics of patients and normal control subjects.a

Sex, M/F Age (yrs) BMI (kg/m2) Duration of illness (yrs) Smokers Smoked cigarettes

Risperidone (n ¼ 41)

Haloperidol (n ¼ 37)

Control subjects (n ¼ 30)

30/11 43.8  6.4 24.4  4.6 21.6  10.9 28 (68%) 12.4  8.3

30/7 43.7  8.1 24.5  4.4 19.2  9.4 24 (65%) 12.1  8.1

22/8 40.4  10.3 24.6  4.9 NA 19 (63%) 11.9  5.0

a There were no significant differences among risperidone, haloperidol and control groups on any characteristic by c2 test and analysis of variance (ANOVA), followed by post hoc tests (Fisher’s LSD test).

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X.Y. Zhang et al. / Neuropharmacology 62 (2012) 1928e1934

were maintained for the full 12 weeks. The antipsychotics were orally administered, and there were special research nurses who supervised the patients to take medication. Four clinical psychiatrists who were blind to treatment conditions assessed the Positive and Negative Symptom Scales (PANSS) (Kay et al., 1987) at baseline and at post-treatment. Baseline was at the start of the randomized intervention. The Treatment Emergent Symptom Scale (TESS) was used by a psychiatrist to measure the side-effect in all patients after 12 weeks of treatment. To ensure consistency and reliability of rating across the study, these four raters simultaneously attended a training session in the use of the PANSS before the start of the study. After training, repeated assessments during the course of the study showed that a correlation coefficient greater than 0.8 was maintained for the PANSS total score.

2.3. Measurement of whole blood SOD and NO levels Samples of patients were collected between 7 and 9 a.m. following an overnight fast at the end of the 2-week washout period and after 12-weeks of treatment. Samples from healthy controls were collected during this same period as that of the patients. Twenty microliters of whole blood were placed in 2 ml of double-distilled water and shaken sufficiently. The hemoglobin was measured, and the other hemolytic samples were stored at 70  C until assessment. 2.3.1. SOD measurement A technician who was blind to the diagnostic status of subjects evaluated the whole blood SOD levels using a commercially available radioimmunoassay (RIA; Ban-Ding Biomedical, Inc., Chinese Academy of Sciences, Beijing, China) within 1 month of storage. A full description of the assays has been given in our previous report (Zhang et al., 2003c). The sensitivity was 2 ng/mL, and the inter-assay and intra-assay variability was 6% and 8%, respectively. 2.3.2. NO assessment A technician who was blind to the diagnostic status of subjects evaluated the plasma NO oxidative metabolites using the Griess method as the nitrite concentration after nitrate reduction to nitrite (Fiddler, 1977). Briefly, 50 mL of 1% sulfanilamide was added to the samples, incubated for 5e10 min, and then 50 mL of 0.1% N-1-naphthylethylenediamine dihydrochloride was added. The reaction was incubated at room temperature for 5e10 min, and absorbance at 540 nm was measured, using sodium nitrite solution as standard. The sensitivity was 2 mmol/L, and the inter-assay and intra-assay variability was 7% and 9%, respectively. Levels of plasma NO are reported in mmol/L. 2.4. Statistical analyses Demographic characteristics of the risperidone, haloperidol, and control groups were compared using chi squared test or Fisher exact test for categorical variables and ANOVA for continuous variables. The principal outcome analysis consisted of analyses of covariance (ANCOVA) for PANSS and its subscales with betweentreatment comparisons of the changes from baseline scores to the end of 12 weeks of treatment, with baseline ratings as the covariates. Since both SOD and NO levels were normally distributed in patients and normal controls, a parametric test had been used. Analysis of covariance (ANCOVA) was performed, using baseline SOD or NO levels as covariates, followed by Fisher’s least significant difference (LSD) test to compare the differences in SOD or NO levels between the groups. Correlation among SOD, NO levels and clinical ratings were examined by Pearson correlation coefficients, and where significant, the Bonferroni correction was used. Total PANSS scores and its sub-scores, SOD, and NO were examined by multivariate regression analyses. All statistical tests were two-tailed and were considered to be statistically significant at p < 0.05.

3. Results 3.1. Demographic data Table 1 shows characteristics of the patients and normal controls. There was no significant difference between patient and normal control groups on any characteristic, such as sex, age, body mass index (BMI) and smoking. No significant relationships between age, sex, smoking or BMI and blood SOD or NO were noted either for the whole group, or when the normal controls and patients were examined separately (all p > 0.05). Age of onset of psychosis, duration of illness, and hospitalization did not significantly correlate with SOD or NO levels in the patient group (all p > 0.05).

Five patients dropped out during the course of study: one on risperidone, and 4 on haloperidol (Zhang et al., 2001). Briefly, both drugs improved PANSS total scores and all subscore measures (p < 0.01e0.001) with risperidone showing greater improvement on PANSS total score (F ¼ 4.98, df ¼ 1,70, p ¼ 0.03), the general psychopathology subscore (F ¼ 6.92m, df ¼ 1,70, p < 0.01), and the negative symptom subscore (F ¼ 3.57, df ¼ 1,70, p ¼ 0.056), but not the positive symptom subscore (F ¼ 2.15, df ¼ 1,70, p > 0.05) (Table 2). The total score of TESS represents the severity of side-effects. There was a significant difference in total scores between the risperidone group (2.9  3.4), and the haloperidol group (6.9  3.8; F ¼ 8.34, df ¼ 1,70, p < 0.001) (Table 2). After treatment, there was a significant difference in the dose of benzhexol hydrochloride taken by the patients between the risperidone group and haloperidol group (2.6  2.8 mg/day versus 4.4  3.0 mg/day; F ¼ 5.46, df ¼ 1,67, p < 0.02). In addition, there was a trend toward a significant difference between the percentage of patients who required antiparkinsonian medication in the risperidol group (8/38), and in the haloperidol group (22/32) (c2 ¼ 3.243, df ¼ 1, p ¼ 0.07). 3.2. Changes of blood SOD and NO before and after treatment As shown in Table 3, blood SOD levels were significantly lower in the healthy controls (482.9  109.9 ng/mg Hb) than the patients at baseline (772.1 117.7 ng/mg Hb), but not at post-treatment (490.4  87.1 ng/mg Hb), and showed a significant decline after treatment (F ¼ 14.4, df ¼ 2, p < 0.001). There was no significant difference in Hb levels at the baseline and post-treatment (127.1 12.3 g/L vs. 125.6  13.9 g/L; p > 0.05). SOD levels significantly declined from baseline to week 12 for both the risperidone (F ¼ 44.7, df ¼ 1,78, p < 0.001) and haloperidol groups (F ¼ 38.6, df ¼ 1,66, p < 0.001) with no difference in the amount of decline between these two medications. Plasma NO levels were significantly higher in patients at baseline (7.3  3.5 mmol/L) and at post-treatment (7.1  2.6 mmol/L) than in the healthy controls (4.0  2.1 mmol/L) (F ¼ 11.5, df ¼ 2, p < 0.001), and showed no change with treatment (F ¼ 0.23, df ¼ 1,142, p > 0.05). NO levels were not significantly different between baseline and week 12 for both the risperidone (F ¼ 0.53, Table 2 Comparison of scores at baseline and week 12 on the total and subscores of PANSS in risperidone and haloperidol groups. Treatmenta

Week 12

Fb

dfb

pb

PANSS total score Risperidone 82.4  22.4 Haloperidol 79.2  21.7

61.8  20.6*** 64.7  16.6***

4.98

1,70

0.03

P-subscore Risperidone Haloperidol

17.9  7.5 15.3  7.9

12.9  6.5*** 11.4  5.8***

2.15

1,70

0.14

N-subscore Risperidone Haloperidol

26.6  8.7 28.1  5.9

22.1  8.5*** 24.6  5.5**

3.57

1,70

0.056

G-subscore Risperidone Haloperidol

37.9  11.5 36.1  11.8

26.7  8.7*** 28.6  8.5***

6.92

1,70

0.01

8.34

1,70

0.001

TESS total score Risperidone Haloperidol

Baseline

2.9  3.4 6.9  3.8

Indicates comparison between pre- and post-treatment. **p < 0.01, ***p < 0.001. a PANSS ¼ Positive and Negative Syndrome Scale; P-subscore ¼ positive symptom subscore; N-subscore ¼ negative symptoms subscore; G-subscore ¼ general psychopathology subscore. TESS ¼ Treatment Emergent Symptom Scale (TESS). b F, df and p referred to comparison between risperidone and haloperidol groups at week 12.

X.Y. Zhang et al. / Neuropharmacology 62 (2012) 1928e1934

Baseline

Week 12

F

df

P

SOD Risperidone Haloperidol Healthy controls

794.4  125.7*** 749.8  109.7*** 482.9  109.9

498.6  97.8 482.2  76.5

44.7 38.6

1,78 1,66

0.000 0.000

NO Risperidone Haloperidol Healthy controls

7.4  3.4*** 7.1  3.7*** 4.0  2.1

6.9  2.6 7.1  2.5

0.53 0.04

1,78 1,66

0.46 0.95

4000 Blood SOD levels, ng/mg Hb

Table 3 Comparison of SOD, NO levels at baseline and week 12 in risperidone and haloperidol groups.

1931

A

3000

r=0.27 df=74 p=0.014

2000

1000

0 20

60

Indicate comparison to healthy controls. ***p < 0.001.

3.3. Relationship of SOD and NO with clinical outcome Correlation analysis shows no significant relationship between SOD and NO in control group (p > 0.05), or in patient group at baseline and at post-treatment (both p > 0.05), or between change in SOD and change NO during treatment (p > 0.05). However, correlation analysis showed a significant positive relationship between SOD levels and both the PANSS total score (r ¼ 0.27, n ¼ 74, p < 0.05; Fig. 1A) and the PANSS positive symptom subscore (r ¼ 0.24, df ¼ 74, p < 0.05; Fig. 1B). There were no significant relationships between NO and any of clinical symptoms (all p > 0.05). Using the criteria of a 20% or more improvement in PANSS total score ratings for defining “responder” or “non-responder” (Kane et al., 1988), the responder group showed significantly lower SOD levels at baseline (513  125 ng/mg Hb vs 593  125 ng/mg Hb, F ¼ 4.56, df ¼ 69, p < 0.05). The reduction in SOD levels after treatment had a significant correlation with the reduction of PANSS total score (r ¼ 0.31, df ¼ 69, p < 0.05) (Fig. 2A) and the reduction of PANSS negative subscore (r ¼ 0.32, n ¼ 69, p < 0.01) (Fig. 2B). Both of them, however, did not pass through Bonferroni test. Furthermore, the reduction in SOD levels after treatment had no significant correlation with the reduction of PANSS general psychopathological subscore (r ¼ 0.18, n ¼ 69, p ¼ 0.15) (Fig. 2C) and the reduction of PANSS positive subscore (r ¼ 0.22, df ¼ 69, p > 0.05) (Fig. 2D). The improvement in the PANSS total and subscale scores had no association with NO levels (all p > 0.05). In addition, the TESS total score had no association with SOD or NO levels (all p > 0.05). Moreover, anti-cholinergic drugs had no relationship with SOD or NO levels (all p > 0.05). Multiple linear regression was performed to explore the predictors of the clinical outcome. Since there were no differences between the risperidone and haloperidol treatments on SOD and NO, the simultaneous and stepped-down analysis was performed on the combined sample. The treatment effect was defined by the reduction in the PANSS total score from baseline to week 12 as the dependent variable. The covariates included a demographic variable (sex, age and smoking status), clinical variables (age at onset, age at first admission, and duration of illness), and biomarkers (baseline SOD, and NO levels), as the independent variables. The result showed that sex (b ¼ 0.37, t ¼ 2.92, p < 0.01) and baseline SOD levels (b ¼ 0.24, t ¼ 2.27, p < 0.05) were associated with the clinical outcome, which accounted for 21% of the variance (adjusted R2 ¼ 0.21). 4. Discussion The major findings of this study are that (1) blood SOD and NO levels were elevated in drug-free chronic schizophrenic patients

Blood SOD levels, ng/mg Hb

df ¼ 1,78, p ¼ 0.46) and haloperidol groups (F ¼ 0.04, df ¼ 1,66, p ¼ 0.95), with no difference in NO levels at both time points between these two medications (both p > 0.05).

100

140

180

PANSS total score 4000

B

r=0.24 df=74 p=0.045

3000

2000 1000

0 0

10

20

30

40

PANSS positive subscore

Fig. 1. There was a significant positive relationship between SOD levels and both the PANSS total score (r ¼ 0.27, df ¼ 74, p < 0.05) and the PANSS positive symptom subscale score (r ¼ 0.24, df ¼ 74, p < 0.05).

compared with controls; (2) both risperidone and haloperidol may reduce elevated blood SOD levels in schizophrenia, with no significant difference between these two drugs; (3) both risperidone and haloperidol produced no significant influence on the elevated plasma NO in schizophrenia. (4) The blood SOD levels at baseline and the reduction (normalization) of SOD levels during treatment were correlated with improvement in PANSS scores. To our knowledge, this is the first study determining the effects of the typical and the atypical antipsychotics on SOD and NO in patients with schizophrenia from systematic, double-blind clinical trials. Furthermore, we have also explored the relationships between the change in SOD and NO and the improvement in clinical symptoms. Our results suggest that both risperidone and haloperidol may produce similar effects on free radical parameters. High blood SOD levels have been reported previously in schizophrenia (Reddy et al., 1991; Yao et al., 2000; Herken et al., 2001; Kuloglu et al., 2002; Zhang et al., 2003a,b; Gama et al., 2006; Kunz et al., 2008; Padurariu et al., 2010), although some studies have reported low SOD levels in schizophrenic patients (Akyol et al., 2002; Ranjekar et al., 2003; Zhang et al., 2006; Ben Othmen et al., 2008). The high SOD activity may be induced in response to increased oxidative tone and abnormally high levels of free radicals in schizophrenia (Lohr et al., 2003; Lee and Kim, 2008). Abnormally high NO levels in schizophrenic patients in our present study are consistent with most but not all previous studies (Das et al., 1995, 1998; Herken et al., 2001; Taneli et al., 2004). NO itself is a free radical and can also produce hydroxyl and nitrogen dioxide radicals that are highly diffusible and widely distributed in various regions of the brain (Yermolaieva et al., 2000). Since NO directly interacts with NMDA receptors, it might contribute to the pathogenesis of schizophrenia through glutamate neurotransmission (Bernstein et al., 2005; Lee and Kim, 2008). Therefore, elevated levels of SOD and NO in schizophrenic patients suggest that oxidation mechanisms may be involved in the pathogenesis of schizophrenia (Yao et al., 2001, 2004).

X.Y. Zhang et al. / Neuropharmacology 62 (2012) 1928e1934

r=0.31 df=69 p<0.05

A

2500

Blood SOD change (ng/mg Hb)

Blood SOD change (ng/mg Hb)

1932

1500

500

-500 -20

30

2500

B

1500

500

-500

-1 0

80

10

1500

500

-500

-5

10

D

2500

r=0.22 df=69 p>0.05

Blood SOD change (ng/mg Hb)

Blood SOD change (ng/mg Hb)

C

30

PANSS negative subscore reduction

PANSS total score reduction

2500

r=0.33 df=69 p<0.01

25

PANSS positive subscore reduction

r=0.18 df=69 p>0.05

1500

500

-500

-15

15

45

PANSS general subscore reduction

Fig. 2. The reduction in SOD levels after treatment had a significant correlation with the reduction of PANSS total (A) or negative subscale score (B), but no significant correlation with the reduction of PANSS general psychopathological subscale (C) or positive subscale score (D).

The second finding of our study showed that both the typical antipsychotic haloperidol and atypical antipsychotic risperidone equally reduced the initially high blood SOD levels in schizophrenia over 12 weeks. These results are compatible with Yao’s study of SOD and GSH-Px using haloperidol in a within-subject, repeated-measures, on-off treatment design (Yao et al., 1998). We also replicated our previous finding that long-term treatment with both typical and atypical antipsychotics may produce the similar effects on the activities of the antioxidant enzymes SOD, GSH-Px and CAT, as well as the levels of lipid peroxidation malondialdehyde (MDA) (Zhang et al., 2006). Hence, it seems that both typical and atypical antipsychotic drugs may have a similar regulatory effect on SOD. The similar effects of risperidone and haloperidol on SOD are probably due to their similar blockade of dopamine (DA) hyperactivity in schizophrenia. Neuroimaging studies have revealed dosedependent dopamine D2 receptor occupancies during treatment with haloperidol of 67e94% for a dose of 5e20 mg/d, and risperidone occupies D2 receptors between 63% and 89% at doses of 2e12 mg/d (Schotte et al., 1996). Hence, the similar high D2 receptor occupancies may occur with the doses of risperidone and haloperidol used in the present study. The third finding was that neither risperidone nor haloperidol reduced the elevated plasma NO in schizophrenia. These negative findings agree with previous findings that atypical antipsychotics had no effect on neuronal NOS in rat brain, or on serum NO metabolites in schizophrenia patients (Tarazi et al., 2002). However, a recent study reported that 6 weeks of treatment with risperidone partially normalized low NO metabolite levels seen at baseline, and that treatment responders showed significantly increased NO metabolite levels (Lee and Kim, 2008). Clinical status of patients (acute vs. chronic, active phase vs. remission), ethnicity, illness course, and different techniques used to measure NO levels might be responsible for this discrepancy, and further study, especially in first-episode and drug-naïve patients will be needed.

The fourth major findings of this study are that the responders based on the PANSS showed lower SOD levels at baseline, and change in SOD during treatment was related to clinical outcome. These results also indicate that poor responders presented more abnormal free radical parameters and that blood SOD levels may be useful for predicting the antipsychotic response. Taken together, these findings indicated that there may be a close relationship between SOD and symptoms of schizophrenic patients, and we speculated that alteration of the SOD level may be a consequence of changed symptoms in schizophrenic patients (Zhang et al., 2003a,b). However, considering that the correlations between change in SOD and improvement of PANSS scores were only marginally significant, and did not pass through Bonferroni test, these findings are considered preliminary, and need to be substantiated in the larger sample size, or in the first-episode and drug naïve schizophrenic patients. Several limitations of the present study should be noted here. First, SOD levels were measured by RIA, which has not been verified for accuracy in comparison with other currently published SOD biochemical assay procedures. Differences in techniques of measuring SOD may have accounted for some discrepancies. Second, the possible effects of antipsychotic treatment before the washout period may play a role. Our patients had undergone chronic long-term treatment with antipsychotics, mainly typical antipsychotics. Studies have shown that the erythrocyte SOD is high in patients medicated with primarily typical antipsychotics (Reddy et al., 2003). Thus, the 2-week washout may not have been adequate for the erythrocyte enzymes to change, because the halflife of the erythrocytes is 120 days. Therefore, it is likely that after drug withdrawal, erythrocyte SOD levels start declining, and the decline found here is probably a result of withdrawal of previous medication and not just the result of antipsychotic treatment. Third, the double-blind study was the original risperidone/haloperidol comparison done nearly a decade ago (Zhang et al., 2001); however, the blood samples from that study were recently utilized

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for the current study. Although this is not uncommon practice, this raises a methodological issue e sample storage time, and its potential effects on SOD and NO. It is known that free radicals are notoriously active and storage has been shown to be an issue. Hence, the possible effects of sample storage time could not be ruled out. We are planning to examine SOD and NO levels in samples from different storage time points, such as 1 month, 3 months, 6 months, 1 year, two years, 5 years and 10 years in an ongoing study to confirm this issue. Fourth, SOD, GSH-Px, and CAT act cooperatively and sequentially in vivo to provide synergistic protection against oxidative damage and in the present study, only SOD was examined, which probably could not reveal in detail the observed findings of antioxidant abnormalities in schizophrenia. Fifth, it is worthy of mentioning that the baseline levels or changes in SOD or NO levels may have no effect on oxidative stress since no measure of oxidative stress, such as lipid peroxidation, was assessed in this study. Sixth, NO is biosynthesized endogenously from L-arginine and oxygen by various nitric oxide synthase (NOS) enzymes. In blood, endothelium-derived NO plays a crucial role in vascular function and homeostasis. Blood NO production is almost exclusively attributable to the activity of endothelial NOS (eNOS) (Bernstein et al., 2005). However, only blood NO was measured in our present study, which does not show fully the NO signaling pathway in blood. Hence, eNOS deserves further investigation in the future studies. In the present study, there are three main reasons that we did not measure eNOS. First, while endothelium-derived NO has been identified as a major player in stroke and ischemia, its role in neuropsychiatric disorders is less clear (Bernstein et al., 2005). Second, there has been no previous study to report the result of eNOS in schizophrenia. In line with previous studies (Akyol et al., 2002; Herken et al., 2001), we focused on NO levels and not eNOS levels. Third, there was no a commercially available kit for eNOS in plasma when this study was conducted. However, we are planning to examine eNOS activity in an ongoing study, including a large group of first-episode and drug-naïve patients with schizophrenia, as well as a group of sex-, age- and smoking-matched healthy controls. In summary, blood SOD levels and plasma NO levels were significantly higher in drug-free patients with chronic schizophrenia, suggesting that oxidative stress may be implicated in the pathophysiology of schizophrenia. Both haloperidol and risperidone significantly decreased the initially higher blood SOD levels in schizophrenic patients, suggesting that both typical and atypical antipsychotic drugs may at least partially normalize abnormal free radical metabolism in schizophrenia, with the similar effects on the parameters of oxidative stress. The negative findings concerning the effects of risperidone and haloperidol on the elevated plasma NO levels suggest that plasma NO levels may serve as a trait mark for a subgroup of schizophrenia patients. Patients who are responders to antipsychotic treatments displayed comparatively “normalized” SOD levels at baseline, and greater change in SOD was correlated with greater symptom improvement, suggesting that blood SOD levels may be useful for predicting the antipsychotic response of schizophrenia patients. These data may help better use of antipsychotics and augmentation strategies with antioxidants in the treatment of schizophrenia. Acknowledgment This study was partially funded by the Stanley Medical Research Institute (03T-459 and 05T-726), the Beijing Municipal Natural Science Foundation (ID: 7072035), and the Department of Veterans Affairs, VISN 16, Mental Illness Research, Education and Clinical

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