Haloperidol treatment induces tissue- and sex-specific changes in DNA methylation: a control study using rats
© Shimabukuro et al; licensee BioMed Central Ltd. 2006
Received: 06 November 2006
Accepted: 29 November 2006
Published: 29 November 2006
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.
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.
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).
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.
Epidemiological studies have established that genetic factors play a major role in the development of schizophrenia. However, the discordance rate for schizophrenia between monozygotic twins is approximately 50%, suggesting that epigenetic and/or environmental factors are also involved in the development of the disease. Despite extensive research, the molecular etiology of schizophrenia remains enigmatic [1, 2]. The use of microarrays or DNA chips for genome-wide analysis of gene expression is showing that the underlying variation in gene expression among individuals may contribute to the development of complex traits and characteristics [3, 4]. Epigenetic modifications, such as DNA methylation, play an important role in the regulation of gene expression, primarily through their role in regulating chromatin structure and function . Defects in epigenetic factors are linked to several diseases [6, 7]. For example, Rett syndrome, a neurodevelopmental disorder, is caused by mutations in the gene encoding methyl-CpG-binding protein-2 (MECP2) , and a lpha-t halassemia/mental r etardation X-linked (ATRX) syndrome is caused by mutations in ATRX, which encodes a member of the SWI/SNF family of chromatin remodeling proteins. Patients with ATRX syndrome exhibit severe mental retardation as well as alpha-thalassemia .
Unlike Rett and ATRX syndromes, symptoms of schizophrenia appear later in life, suggesting that environmental factors contribute to the development of the disease. The methylation state of the genome undergoes highly dynamic changes, extensive demethylation and reconstruction during early embryogenesis, yet once established, is very stable . Nevertheless, some epigenetic signals, including DNA methylation, can be transmitted from one generation to the next, and are influenced by environmental or intrinsic biological factors [11–14]. Thus, DNA methylation and/or other epigenetic modifications of the genome may help explain the ambiguity of inherited schizophrenia and the role, if any, of environmental factors in the etiology of the disease.
In developing a model of schizophrenia , we examined global methylation of peripheral leukocyte DNA from more than 200 patients with schizophrenia, and compared it to global methylation in healthy subjects. The results revealed lower mC content in male patients than in male controls, although the difference did not reach a statistically significance . In the present study, we were interested in determining whether this difference was a secondary effect of anti-psychotic medications.
Animals and chemical
Eight-week-old male and female Sprague-Dawley rats (330 – 360 g and 190 – 230 g, respectively) were purchased from Kyudo Co., Ltd. (Kumamoto, Japan), housed in standard cages in a controlled environment at a constant room temperature (24 ± 2°C) and humidity (50–70%), and maintained on a 12 h light-dark cycle. Food and water were provided ad libitum through out the experiment. Haloperidol was kindly provided by Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). The bulk powdered chemical was dissolved in 0.1 M glacial acetic acid buffered to pH6.0 with NaOH at concentration of 0.25 mg/ml . All animal experiments were reviewed and approved by the Ryukyu University Animal Welfare Committee.
Ten male and 10 female rats were administered 0.5 mg/kg haloperidol intraperitoneally on a daily basis . Control rats received the same volume of vehicle solution alone under parallel conditions. After 21 days of daily injection, the rats were sacrificed by injection of sodium pentobarbital (120 mg/kg) intraperitoneally. Trunk blood was collected using a heparinized syringe, and plasma was prepared by centrifugation at 2,000 rpm for 10 min, then stored at -30°C until use. Layered cells, which include whole white blood cells, following centrifugation were isolated and set aside for DNA extraction. Whole brains, excluding the cerebellum and olfactory bulb, liver, and kidney were rapidly excised, rinsed twice in saline, then snap-frozen in liquid nitrogen.
mC content in blood leukocyte DNA was evaluated using high performance liquid chromatography (HPLC). All reagents and conditions for HPLC analysis have been described previously . Global methylation levels are expressed as the ratio of mC to the sum of 2'-deoxycytidine and mC (%).
Statistical analyses were performed using Stat View software (SAS Institute Inc., Cary, NC, USA). Differences in mean mC content between the two groups were evaluated with the Mann-Whitney U test. Results were considered statistically significant when the P value was less than 0.05.
Physiological state of haloperidol-treated rats
Body weight and plasma concentrations of prolactin and haloperidol for control and haloperidol-treated rats
Body weight (g) at
Plasma concentrations of
351.5 ± 6.9
476.0 ± 8.4
64.7 ± 43.7
4.3 ± 0.9b
342.5 ± 6.3
473.0 ± 9.5
37.3 ± 10.0b
1.5, 2.0, <1.3c
211.0 ± 15.2
298.0 ± 16.9
441.1 ± 243.3
211.0 ± 12.9
300.0 ± 15.6
150.5 ± 112.7b
Sex- and tissue- specific global methylation levels in control rats
Tissue- and sex-specificity of methylcytosine (mC) content (%) in control rats
mC content in leukocyte, brain, and liver DNA (mean ± SD)
3.843 ± 0.042
3.650 ± 0.023
3.412 ± 0.033
3.771 ± 0.048
3.652 ± 0.031
3.426 ± 0.027
Effect of haloperidol on global methylation levels in leukocytes, brain, and liver
Effects of haloperidol on the mC content of leukocyte, brain, and liver DNA
mC content (mean ± SD)
3.817 ± 0.036
3.632 ± 0.047
3.445 ± 0.027
3.843 ± 0.042
3.650 ± 0.023
3.412 ± 0.033
3.812 ± 0.050
3.615 ± 0.031
3.449 ± 0.055
3.771 ± 0.048
3.652 ± 0.031
3.426 ± 0.027
The aim of the current study was to determine whether global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia was a secondary effect of their medication. As a model system for studying this question, we chose haloperidol-injected rats, since haloperidol, until recently, was a commonly used drug for the treatment of schizophrenia. In leukocyte DNA from control rats that were injected with buffer alone, there was a sex-dependent difference in mC content (females lower than males), similar to what was previously observed in humans [16, 19] The apparent paradox of a lower methylation state in females may be partly attributed to global hypomethylation of the inactive X chromosome . The global methylation level in rat liver and brain was also consistent with what has been observed in humans: liver < brain . Thus, the data obtained from rats and humans are comparable. In male rats injected with haloperidol, the amount of mC in leukocyte DNA was less than that seen in control male rats, but the difference was not statistically significant. Although further study is needed in order to fully understand the difference in mC content of peripheral leukocyte DNA between male patients with schizophrenia and healthy male subjects, the results of the current study present several interesting and novel findings, and implicate a role for haloperidol, and perhaps other antipsychotic medications, in the alteration of DNA methylation in schizophrenic male patients.
Haloperidol-treatment resulted in a decrease in the amount of mC in leukocyte DNA in male rats, although the difference did not reach a statistical significance. In contrast, we found a trend toward higher levels of mC in leukocyte DNA in female rats treated with haloperidol (P = 0.064). Although the reasons why haloperidol treatment would have opposite effects on global methylation in male and female rats are not clear from this study, it is possible that these differences are the result of disruptions in the balance of hormones in these animals. This is supported by observations that there are sex-dependent differences in the immune response to certain hormones, and in the frequency of occurrence of some autoimmune diseases [21–23]. It is also possible that the observed effect might be mediated by the alteration in the subset profile of white blood cells. The partial discrepancy between the results obtained from patients with schizophrenia and from rats injected with haloperidol will require additional study, in particular into the differences between humans and rodents in the regulation of epigenetic factors, or other systems that may effect the course of disease [24, 25].
In the liver, haloperidol increased global DNA methylation in both males and females, and the difference was statistically significant in males (P = 0.013). In the brain, haloperidol treatment resulted in a decrease in females, and the decrease was statistically significant (P = 0.026). The differential effect of haloperidol on DNA methylation in various tissues suggests that the regulation of global DNA methylation by this drug occurs through multiple, indirect pathways. In these pathways, sex-specific hormones may play a role in modifying the methylation state of DNA, since haloperidol also disturbs the normal regulation of sex-hormone secretion.
Several studies have established that there are gender differences among patients with psychiatric disorders, suggesting that sex-specific hormones play a role in the pathogenesis of these disorders, including schizophrenia [26–29]. It remains a topic of considerable interest whether and how these hormones are involved in disease progression. Clinical phenotypes are most frequently the manifestation of multiple alterations in genetic and environmental factors. In the current study, using a limited number of rats, we were unable to demonstrate a causative effect of haloperidol on the observed hypomethylation of leukocyte DNA in male patients with schizophrenia . However, we did uncover several sex- and tissue-specific effects of haloperidol on DNA methylation. Of note, valproate, a drug that is commonly used to treat bipolar disorder, has been shown to have a demethylating effect . Clarification of the cellular pathways that mediate haloperidol's effect on DNA methylation state will help elucidate the molecular etiologies of schizophrenia.
In the current study, using a limited number of rats, we were unable to demonstrate a causative effect of haloperidol on the observed hypomethylation of leukocyte DNA in male patients with schizophrenia. However, we did uncover several sex- and tissue-specific effects of haloperidol on DNA methylation, which helps the researchers interpret the data in epigenetic studies in schizophrenia, and also would shed light on the mechanism of action of antipsychotics.
We thank J. Sugimoto, H. Shen, Y. Eguchi, T. Oda, and E. Miyajima for their help. This study was supported in part by a Grant-in-Aid for Science Research (A) (No. 13307027) and by a Health and Labour Sciences Research Grant (No. 17230601).
- Owen MJ, Cardno AG, O'Donovan MC: Psychiatric genetics: back to the future. Mol Psychiatry. 2000, 5: 22-31. 10.1038/sj.mp.4000702.View ArticlePubMed
- Baron M: Genetics of schizophrenia and the new millennium: progress and pitfalls. Am J Hum Genet. 2001, 68: 299-312. 10.1086/318212.PubMed CentralView ArticlePubMed
- Cheung VG, Conlin LK, Weber TM, Arcaro M, Jen KY, Morley M, Spielman RS: Natural variation in human gene expression assessed in lymphoblastoid cells. Nat Genet. 2003, 33: 422-425. 10.1038/ng1094.View ArticlePubMed
- Radich JP, Mao M, Stepaniants S, Biery M, Castle J, Ward T, Schimmack G, Kobayashi S, Carleton M, Lampe J, Linsley PS: Individual-specific variation of gene expression in peripheral blood leukocytes. Genomics. 2004, 83: 980-988. 10.1016/j.ygeno.2003.12.013.View ArticlePubMed
- Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003, 33 (Suppl): 245-254. 10.1038/ng1089.View ArticlePubMed
- Jones PA, Laird PW: Cancer epigenetics comes of age. Nat Genet. 1999, 21: 163-167. 10.1038/5947.View ArticlePubMed
- Robertson KD, Wolffe AP: DNA methylation in health and disease. Nat Rev Genet. 2000, 1: 11-19. 10.1038/35049533.View ArticlePubMed
- Shahbazian MD, Zoghbi HY: Rett syndrome and MeCP2: linking epigenetics and neuronal function. Am J Hum Genet. 2002, 71: 1259-1272. 10.1086/345360.PubMed CentralView ArticlePubMed
- Gibbons RJ, McDowell TL, Raman S, O'Rourke DM, Garrick D, Ayyub H, Higgs DR: Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet. 2000, 24: 368-371. 10.1038/74191.View ArticlePubMed
- Hsieh C-L: Dynamics of DNA methylation pattern. Curr Opin Genet Dev. 2000, 10: 224-228. 10.1016/S0959-437X(00)00064-2.View ArticlePubMed
- Roemer I, Reik W, Dean W, Klose J: Epigenetic inheritance in the mouse. Curr Biol. 1997, 7: 277-280. 10.1016/S0960-9822(06)00124-2.View ArticlePubMed
- Morgan HD, Sutherland HG, Martin DI, Whitelaw E: Epigenetic inheritance at the agouti locus in the mouse. Nat Genet. 1999, 23: 314-318. 10.1038/15490.View ArticlePubMed
- Sutherland HG, Kearns M, Morgan HD, Headley AP, Morris C, Martin DI, Whitelaw E: Reactivation of heritably silenced gene expression in mice. Mamm Genome. 2000, 11: 347-355. 10.1007/s003350010066.View ArticlePubMed
- Rakyan VK, Blewitt ME, Druker R, Preis JI, Whitelaw E: Metastable epialleles in mammals. Trends Genet. 2002, 18: 348-351. 10.1016/S0168-9525(02)02709-9.View ArticlePubMed
- Nakamura A, Okazaki Y, Sugimoto J, Oda T, Jinno Y: Human endogenous retroviruses with transcriptional potential in the brain. J Hum Genet. 2003, 48: 575-581. 10.1007/s10038-003-0081-8.View ArticlePubMed
- Shimabukuro M, Jinno Y, Sasaki T, Imamura A, Tsujita T, Fuke C, Umekage T, Tochigi M, Hiramatsu K, Miyazaki T, Oda T, Sugimoto J, Jinno Y, Okazaki Y: Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia: a potential link between epigenetics and schizophrenia. J Psychiatr Res.
- Pehek EA: Comparison of effects of haloperidol administration on amphetamine-stimulated dopamine release in the rat medial prefrontal cortex and dorsal striatum. J Pharmacol Exp Ther. 1999, 289: 14-23.PubMed
- Lynch MR, Woo J: Enhanced haloperidol-induced prolactinstimulation with chronic neuroleptic treatment in the rat. Life Sci. 1991, 49: 1721-1729. 10.1016/0024-3205(91)90314-2.View ArticlePubMed
- Fuke C, Shimabukuro M, Petronis A, Sugimoto J, Oda T, Miura K, Miyazaki T, Ogura C, Okazaki Y, Jinno Y: Age related changes of the 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study. Ann Hum Genet. 2004, 68: 196-204. 10.1046/j.1529-8817.2004.00081.x.View ArticlePubMed
- Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D: Chromosome-wide and promoter-specific analysesidentify sites of differential DNA methylation in normal andtransformed human cells. Nat Genet. 2005, 37: 853-862. 10.1038/ng1598.View ArticlePubMed
- Bouman A, Schipper M, Heineman MJ, Faas MM: Gender difference in the non-specific and specific immune response in humans. Am J Reprod Immunol. 2004, 52: 19-26. 10.1111/j.1600-0897.2004.00177.x.View ArticlePubMed
- Fairweather D, Rose NR: Women and autoimmune diseases. Emerg Infect Dis. 2004, 10: 2005-2011.PubMed CentralView ArticlePubMed
- Grimaldi CM, Hill L, Xu X, Peeva E, Diamond B: Hormonal modulation of B cell development and repertoire selection. Mol Immunol. 2005, 42: 811-820. 10.1016/j.molimm.2004.05.014.View ArticlePubMed
- Ferguson-Smith AC, Sasaki H, Cattanach BM, Surani MA: Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature. 1993, 362: 751-755. 10.1038/362751a0.View ArticlePubMed
- Jinno Y, Ikeda Y, Yun K, Maw M, Masuzaki H, Fukuda H, Inuzuka K, Fujishita A, Ohtani Y, Okimoto T, Ishimaru T, Niikawa N: Establishment of functional imprinting of the H19 gene in human developing placentae. Nat Genet. 1995, 10: 318-324. 10.1038/ng0795-318.View ArticlePubMed
- Szymanski S, Lieberman JA, Alvir JM, Mayerhoff D, Loebel A, Geisler S, Chakos M, Koreen A, Jody D, Kane J: Gender differences in onset of illness, treatment response, course, and biologic indexes in first-episode schizophrenic patients. Am J Psychiatry. 1995, 152: 698-703.View ArticlePubMed
- Rao ML, Kolsch H: Effects of estrogen on brain development and neuroprotection – implications for negative symptoms in schizophrenia. Psychoneuroendocrinology. 2003, 28: 83-96. 10.1016/S0306-4530(02)00126-9.View ArticlePubMed
- Gogos A, Van den Buuse M: Estrogen and progesterone prevent disruption of prepulse inhibition by the serotonin-1A receptor agonist 8-hydroxy-2-dipropylaminotetralin. J Pharmacol Exp Ther. 2004, 309: 267-274. 10.1124/jpet.103.061432.View ArticlePubMed
- Huber TJ, Borsutzky M, Schneider U, Emrich HM: Psychotic disorders and gonadal function: evidence supporting the oestrogen hypothesis. Acta Psychiatr Scand. 2004, 109: 269-274. 10.1046/j.1600-0447.2003.00251.x.View ArticlePubMed
- Detich N, Bovenzi V, Szyf M: Valproate induces replication-independent active DNA demethylation. J Biol Chem. 2003, 278: 27586-27592. 10.1074/jbc.M303740200.View ArticlePubMed
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