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Background: We previously found that there is a subtle difference in the global methylation state of blood leukocyte DNA between male subjects with and without schizophrenia. The aim of the current study was to determine whether this difference was a primary effect of the disease state, or a secondary effect of antipsychotics administered to these patients. Methods: We examined the methyl cytosine (mC) content of DNA from the leukocytes, brain, and liver of rats using high performance liquid chromatography. A total of 40 male and female rats received for 21 days daily injection of haloperidol or vehicle solution alone. Results: In control rats injected with buffer only, there was a sex-dependent difference in mC content in leukocyte DNA (male > female; P = 0.028, n = 10), similar to our previous observations in human peripheral leukocytes. No difference in mC content between the sexes was observed in the brain or liver in buffer-treated animals. Haloperidol treatment slightly decreased the mC content of leukocytes in male rats, but unexpectedly, increased the mC content of leukocytes in females. We observed a trend toward a higher level of mC in the liver in both sexes following haloperidol treatment, compared to buffer-treated animals. In contrast, haloperidol treatment resulted in a decrease in mC content in the brain in females, and this difference was statistically significant (P = 0.026). Conclusion: These results indicate that haloperidol can affect DNA methylation states in the brain, as well as in certain other tissues, and raise the possibility that antipsychotic drugs play a role in the observed disparity in mC content in male subjects with and without schizophrenia. Background extensive research, the molecular etiology of schizophre- Epidemiological studies have established that genetic fac- nia remains enigmatic [1,2]. The use of microarrays or tors play a major role in the development of schizophre- DNA chips for genome-wide analysis of gene expression is nia. However, the discordance rate for schizophrenia showing that the underlying variation in gene expression between monozygotic twins is approximately 50%, sug- among individuals may contribute to the development of gesting that epigenetic and/or environmental factors are complex traits and characteristics [3,4]. Epigenetic modi- also involved in the development of the disease. Despite fications, such as DNA methylation, play an important Page 1 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:37 http://www.behavioralandbrainfunctions.com/content/2/1/37 role in the regulation of gene expression, primarily Experimental procedure through their role in regulating chromatin structure and Ten male and 10 female rats were administered 0.5 mg/kg function . Defects in epigenetic factors are linked to haloperidol intraperitoneally on a daily basis . Con- several diseases [6,7]. For example, Rett syndrome, a neu- trol rats received the same volume of vehicle solution rodevelopmental disorder, is caused by mutations in the alone under parallel conditions. After 21 days of daily gene encoding methyl-CpG-binding protein-2 (MECP2) injection, the rats were sacrificed by injection of sodium , and alpha-thalassemia/mental retardation X-linked pentobarbital (120 mg/kg) intraperitoneally. Trunk blood (ATRX) syndrome is caused by mutations in ATRX, which was collected using a heparinized syringe, and plasma was encodes a member of the SWI/SNF family of chromatin prepared by centrifugation at 2,000 rpm for 10 min, then remodeling proteins. Patients with ATRX syndrome stored at -30°C until use. Layered cells, which include exhibit severe mental retardation as well as alpha-tha- whole white blood cells, following centrifugation were lassemia . isolated and set aside for DNA extraction. Whole brains, excluding the cerebellum and olfactory bulb, liver, and Unlike Rett and ATRX syndromes, symptoms of schizo- kidney were rapidly excised, rinsed twice in saline, then phrenia appear later in life, suggesting that environmental snap-frozen in liquid nitrogen. factors contribute to the development of the disease. The methylation state of the genome undergoes highly HPLC analysis dynamic changes, extensive demethylation and recon- mC content in blood leukocyte DNA was evaluated using struction during early embryogenesis, yet once estab- high performance liquid chromatography (HPLC). All lished, is very stable . Nevertheless, some epigenetic reagents and conditions for HPLC analysis have been signals, including DNA methylation, can be transmitted described previously . Global methylation levels are from one generation to the next, and are influenced by expressed as the ratio of mC to the sum of 2'-deoxycyti- environmental or intrinsic biological factors [11-14]. dine and mC (%). Thus, DNA methylation and/or other epigenetic modifi- cations of the genome may help explain the ambiguity of Statistical analysis inherited schizophrenia and the role, if any, of environ- Statistical analyses were performed using Stat View soft- ware (SAS Institute Inc., Cary, NC, USA). Differences in mental factors in the etiology of the disease. mean mC content between the two groups were evaluated In developing a model of schizophrenia , we exam- with the Mann-Whitney U test. Results were considered ined global methylation of peripheral leukocyte DNA statistically significant when the P value was less than from more than 200 patients with schizophrenia, and 0.05. compared it to global methylation in healthy subjects. The results revealed lower mC content in male patients Results than in male controls, although the difference did not Physiological state of haloperidol-treated rats reach a statistically significance . In the present study, All rats treated with haloperidol grew as well as control we were interested in determining whether this difference rats and did not exhibit noticeable abnormal behavior was a secondary effect of anti-psychotic medications. during the course of the experiment. Body weights and blood concentrations of haloperidol and prolactin at the Methods time the animals were sacrificed are summarized in Table Animals and chemical 1. Eight-week-old male and female Sprague-Dawley rats (330 – 360 g and 190 – 230 g, respectively) were pur- Sex- and tissue- specific global methylation levels in control rats chased from Kyudo Co., Ltd. (Kumamoto, Japan), housed in standard cages in a controlled environment at a con- In a previous study, we found a sex-dependent difference stant room temperature (24 ± 2°C) and humidity (50– in the mC content of human leukocyte DNA, with the 70%), and maintained on a 12 h light-dark cycle. Food level in males being greater than that in females [16,19]. and water were provided ad libitum through out the exper- In the current study, we first compared the mC content of iment. Haloperidol was kindly provided by Dainippon leukocyte DNA in male and female control rats. There was Sumitomo Pharma Co., Ltd. (Osaka, Japan). The bulk a statistically significant difference in the mC content of powdered chemical was dissolved in 0.1 M glacial acetic leukocyte DNA (males greater than females), which was acid buffered to pH6.0 with NaOH at concentration of consistent with our previous results in humans. However, 0.25 mg/ml . All animal experiments were reviewed there was no difference in the mC content between males and approved by the Ryukyu University Animal Welfare and females in either the brain or liver DNA (Table 2). The Committee. differences in global methylation levels among different Page 2 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:37 http://www.behavioralandbrainfunctions.com/content/2/1/37 Table 1: Body weight and plasma concentrations of prolactin and haloperidol for control and haloperidol-treated rats Sex Treatment Body weight (g) at Plasma concentrations of beginning end prolactin (ng/ml) haloperidol (ng/ml) Male haloperidol 351.5 ± 6.9 476.0 ± 8.4 64.7 ± 43.7 4.3 ± 0.9 b c placebo 342.5 ± 6.3 473.0 ± 9.5 37.3 ± 10.0 1.5, 2.0, <1.3 Female haloperidol 211.0 ± 15.2 298.0 ± 16.9 441.1 ± 243.3 ND placebo 211.0 ± 12.9 300.0 ± 15.6 150.5 ± 112.7 ND Each group consisted of 10 rats. n = 5 Haloperidol was below the detection limit in three of the five rats examined. ND: not determined due to a high background activity of unknown origin. Plasma concentrations of prolactin and haloperidol were determined by the [ I]-labelled radioimmunoassay and colloidal gold immunoassay, respectively. tissues were quite remarkable (leukocytes > brains > liv- 0.220, p = 0.205 in the liver; ρ = -0.193, p = 0.279 in the ers). brain). Effect of haloperidol on global methylation levels in Discussion leukocytes, brain, and liver The aim of the current study was to determine whether We next compared the mC content in haloperidol- global hypomethylation of peripheral leukocyte DNA in injected rats (drug group) to buffer treated control ani- male patients with schizophrenia was a secondary effect mals (control group) by sex and tissue type. In males, the of their medication. As a model system for studying this mean level of mC content of leukocyte DNA was slightly question, we chose haloperidol-injected rats, since lower in the drug group than in the control group, but the haloperidol, until recently, was a commonly used drug for difference was not statistically significant. In contrast, in the treatment of schizophrenia. In leukocyte DNA from female leukocytes, we observed a trend toward higher mC control rats that were injected with buffer alone, there was levels in the drug group compared to the control group, a sex-dependent difference in mC content (females lower although again, the difference did not reach a statistically than males), similar to what was previously observed in significant level (P = 0.064). Thus, the sex-dependent dif- humans [16,19] The apparent paradox of a lower methyl- ference in mC content of leukocyte DNA between male ation state in females may be partly attributed to global and female rats appeared to be ameliorated by haloperi- hypomethylation of the inactive X chromosome . The dol treatment. Global methylation in the brains of global methylation level in rat liver and brain was also haloperidol-treated rats was lower than in the brains of consistent with what has been observed in humans: liver female control animals, and this difference was statisti- < brain . Thus, the data obtained from rats and cally significant (P = 0.026). There was a higher level of humans are comparable. In male rats injected with methylation of liver DNA in the drug group compared to haloperidol, the amount of mC in leukocyte DNA was less the control group, for both males and females, and in than that seen in control male rats, but the difference was males, this difference was statistically significant (P = not statistically significant. Although further study is 0.013). These results are summarized in Table 3. No sta- needed in order to fully understand the difference in mC tistically significant correlation between prolactin concen- content of peripheral leukocyte DNA between male tration and mC content in any tissue was detected patients with schizophrenia and healthy male subjects, (Spearman's ρ = -0.104, p = 0.570 in leukocytes; ρ = - the results of the current study present several interesting Table 2: Tissue- and sex-specificity of methylcytosine (mC) content (%) in control rats mC content in leukocyte, brain, and liver DNA (mean ± SD) Sex (n) Leukocytes P Brain P Liver P Male (10) 3.843 ± 0.042 0.028 3.650 ± 0.023 0.791 3.412 ± 0.033 0.496 Female (10) 3.771 ± 0.048 3.652 ± 0.031 3.426 ± 0.027 n: numbers of rats analyzed. Differences in mean mC content between male and female rats were evaluated with the Mann-Whitney U test. Page 3 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:37 http://www.behavioralandbrainfunctions.com/content/2/1/37 Table 3: Effects of haloperidol on the mC content of leukocyte, brain, and liver DNA mC content (mean ± SD) Sex Treatment Leukocytes P Brain P Liver P Male Haloperidol 3.817 ± 0.036 0.212 3.632 ± 0.047 0.273 3.445 ± 0.027 0.013 Placebo 3.843 ± 0.042 3.650 ± 0.023 3.412 ± 0.033 Female Haloperidol 3.812 ± 0.050 0.064 3.615 ± 0.031 0.026 3.449 ± 0.055 0.089 Placebo 3.771 ± 0.048 3.652 ± 0.031 3.426 ± 0.027 Each treatment-group consisted of 10 rats. injected with solvent, NaOH-buffered acetic acid (pH6.0), alone. Differences in mean mC content between haloperidol- and placebo-treated groups were evaluated with the Mann-Whitney U test. and novel findings, and implicate a role for haloperidol, Several studies have established that there are gender dif- and perhaps other antipsychotic medications, in the alter- ferences among patients with psychiatric disorders, sug- ation of DNA methylation in schizophrenic male patients. gesting that sex-specific hormones play a role in the pathogenesis of these disorders, including schizophrenia Haloperidol-treatment resulted in a decrease in the [26-29]. It remains a topic of considerable interest amount of mC in leukocyte DNA in male rats, although whether and how these hormones are involved in disease the difference did not reach a statistical significance. In progression. Clinical phenotypes are most frequently the contrast, we found a trend toward higher levels of mC in manifestation of multiple alterations in genetic and envi- leukocyte DNA in female rats treated with haloperidol (P ronmental factors. In the current study, using a limited = 0.064). Although the reasons why haloperidol treat- number of rats, we were unable to demonstrate a causa- ment would have opposite effects on global methylation tive effect of haloperidol on the observed hypomethyla- in male and female rats are not clear from this study, it is tion of leukocyte DNA in male patients with possible that these differences are the result of disruptions schizophrenia . However, we did uncover several sex- in the balance of hormones in these animals. This is sup- and tissue-specific effects of haloperidol on DNA methyl- ported by observations that there are sex-dependent dif- ation. Of note, valproate, a drug that is commonly used to ferences in the immune response to certain hormones, treat bipolar disorder, has been shown to have a demeth- and in the frequency of occurrence of some autoimmune ylating effect . Clarification of the cellular pathways diseases [21-23]. It is also possible that the observed effect that mediate haloperidol's effect on DNA methylation might be mediated by the alteration in the subset profile state will help elucidate the molecular etiologies of schiz- of white blood cells. The partial discrepancy between the ophrenia. results obtained from patients with schizophrenia and from rats injected with haloperidol will require additional Conclusion In the current study, using a limited number of rats, we study, in particular into the differences between humans and rodents in the regulation of epigenetic factors, or were unable to demonstrate a causative effect of haloperi- other systems that may effect the course of disease dol on the observed hypomethylation of leukocyte DNA [24,25]. in male patients with schizophrenia. However, we did uncover several sex- and tissue-specific effects of haloperi- In the liver, haloperidol increased global DNA methyla- dol on DNA methylation, which helps the researchers tion in both males and females, and the difference was interpret the data in epigenetic studies in schizophrenia, statistically significant in males (P = 0.013). In the brain, and also would shed light on the mechanism of action of haloperidol treatment resulted in a decrease in females, antipsychotics. and the decrease was statistically significant (P = 0.026). The differential effect of haloperidol on DNA methylation Acknowledgements We thank J. Sugimoto, H. Shen, Y. Eguchi, T. Oda, and E. Miyajima for their in various tissues suggests that the regulation of global help. This study was supported in part by a Grant-in-Aid for Science DNA methylation by this drug occurs through multiple, Research (A) (No. 13307027) and by a Health and Labour Sciences indirect pathways. In these pathways, sex-specific hor- Research Grant (No. 17230601). mones may play a role in modifying the methylation state of DNA, since haloperidol also disturbs the normal regu- lation of sex-hormone secretion. 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