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Gene expression on liver toxicity induced by administration of haloperidol in rats with severe fatty liver
Legal Medicine 10 (2008) 177–184 www.elsevier.com/locate/legalmed
Gene expression on liver toxicity induced by administration of haloperidol in rats with severe fatty liver Masakazu Hanagama, Hiromasa Inoue *, Munechika Kamiya, Kotaro Shinone, Masayuki Nata Department of Forensic Medicine and Sciences, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu City, Mie 514-8507, Japan Received 21 August 2007; received in revised form 14 December 2007; accepted 26 December 2007 Available online 14 February 2008
Abstract Sudden deaths are often encountered in schizophrenic patients prescribed with antipsychotic drugs, and fatty liver may be more prevalent among patients with schizophrenia. The aim of this study is to investigate the adverse effects of antipsychotic drugs on fatty liver. We administered haloperidol intraperitoneally to fatty liver rats and examined the mRNA expression in the liver. Basic expressions of cytochrome P450 (CYP)1A2, CYP2C11 and CYP3A2 decreased, and response of these CYPs to haloperidol was reduced in the fatty liver. Metabolism of haloperidol was also suppressed in the fatty liver rats. Moreover, hepatic injury by administration of haloperidol was shown pathohistologically and molecular-biologically in severe fatty liver. These results suggest that fatty liver increases susceptibility to adverse effects of haloperidol, possibly leading to life-threatening events. It should be noted by clinicians that excessive dose of antipsychotic drugs may be more harmful in patients with fatty liver. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Schizophrenia; Sudden death; Fatty liver; Haloperidol; Cytochrome P450; Apoptosis
1. Introduction Antipsychotic drugs have been widely used in treatment of schizophrenia [1,2]. In forensic practices, sudden deaths are often encountered in schizophrenic patients, in which it is often difficult to decide the precise causes of death after performing complete autopsy [3]. One of the mechanisms of death might be the occurrence of ventricular arrhythmia and QT prolongation associated with antipsychotic substances, resulting in sudden cardiac death [4–6]; however, it has hardly been investigated whether the effects of these drugs on the other organs contribute to death. Schizophrenic patients are reported to have higher incidence of obesity, diabetes mellitus and dyslipidemia than in the general population [7,8]. These metabolic disorders are closely associated with fatty liver, indicating that fatty liver
*
Corresponding author. Tel./fax: +81 59 231 5014. E-mail address: [email protected] (H. Inoue).
may well be more prevalent among patients with schizophrenia than among the general population, where its occurrence is estimated at 10–34% [9–11]. In forensic medicine, there have been many reports of sudden death cases associated with fatty liver [12–14]. Moreover, it is suggested that drug metabolism may be impaired in patients with fatty liver [15]. However, there have been no reports of investigation into the relationship between the effect of antipsychotic drugs on fatty liver and death. Therefore, this study aimed to investigate the adverse effects of antipsychotic drugs on fatty and normal liver. Haloperidol is one of the most widely used antipsychotic drugs for the treatment of schizophrenia [1]. We administered it to normal rats and fatty liver rats, and from the point of view of drug metabolism and tissue injury, we examined mRNA expression in the liver of cytochrome P450 (CYP)1A2, CYP2C11 and CYP3A2, which are major metabolic enzymes of various drugs in the liver [16–20], and Bcl-2 and Bax, which are anti- and pro-apoptotic proteins [21,22].
1344-6223/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2007.12.006
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M. Hanagama et al. / Legal Medicine 10 (2008) 177–184
2. Materials and methods
2.3. RNA extraction and complementary cDNA synthesis
The experiments were reviewed by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Mie University and carried out under the control of Guidelines for Animal Experiments in the Faculty of Medicine, Mie University and the Law (No. 105) and Notification (No. 6) of the Government of Japan. Moreover, this study followed the ‘‘Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
mRNA was extracted from the liver using QuickGene RNA tissue kit SII (FUJIFILM, Tokyo, Japan) according to the manufacturer’s instructions. Then, this mRNA was utilized as a template, and double-stranded complementary DNA (cDNA) was synthesized by using a first-strand cDNA synthesis kit (ReverTra Ace-a-, Toyobo, Osaka, Japan), according to the attached instructions.
2.4. Polymerase chain reaction quantification of mRNA expression
2.1. Preliminary experiment To induce fatty liver, 3-week-old male Sprague–Dawley (SD) rats (CLEA Japan, Inc., Tokyo, Japan) were reared for 6 weeks with only a special laboratory feed [23]. All the rats were housed in a temperature-controlled room on a 12:12 light–dark cycle. The fatty liver rats (100–170 g) were administered various doses of haloperidol (SerenaceÒ, Dainippon Sumitomo Pharma, Osaka, Japan). When the rats were injected with 40 mg/kg haloperidol intraperitoneally, almost half of them died about 30-min after administration, but the others survived. Accordingly, we adopted 40 mg/kg of haloperidol as an intoxicating dose for rats in this study. 2.2. Animal treatment The experiment was performed to compare two groups: (I) rats with fatty liver as the fatty liver group (50–170 g) and (II) 9- to 12-week-old male SD rats without fatty liver as the normal liver group (210–390 g). Normal liver rats and fatty liver rats were injected with 40 mg/kg haloperidol intraperitoneally. Because almost half of those rats died about 30-min after administration of haloperidol in the preliminary study, survival time for them was regarded as 0.5 h. The surviving rats were killed in a short time in a vacuum chamber at 1, 2 and 4 h after administration of haloperidol. The liver was removed immediately after death, and a portion of it was soaked in RNAlater (Applied Biosystems, Foster City, CA, USA) for one night and then stored at 80 °C for later analysis of mRNA expression. In addition, the other portion of the liver was fixed in 4% formaldehyde, embedded in paraffin, and then 4-lm thick sections were cut for the pathohistological detection of apoptotic cells. We randomly chose the liver samples to analyze from both the fatty liver group (n = 10 for 0.5, 1, 2 and 4 h) and the normal liver group (n = 10 for 0.5, 1, 2 and 4 h). As two pretreatment controls, 9-week-old SD rats without fatty liver (n = 10) and with fatty liver (n = 10), but without administration of haloperidol, were killed and then treated using the same methods.
Primer sequences for cytochrome P450 (CYP)1A2, CYP2C11, CYP3A2, Bcl-2 and Bax cDNA, which were designed using Primer Express version 1.5 (Applied Biosystems, Foster City, CA, USA), are presented in Table 1. The mRNA levels of CYP1A2, CYP2C11, CYP3A2, Bcl-2, Bax and b-actin (an internal reference cDNA) were quantified using a fluorescence detection system (ABI PRISM 7700 sequence detection system, Applied Biosystems), in accordance with the attached instructions. The PCR mixture consisted of 0.5 lM of each primer (CYP1A2, CYP2C11, CYP3A2 Bcl-2 or Bax) and 5 ll of Power SYBR Green Realtime PCR Master Mix (Applied Biosystems) in a final volume of 10 ll. The expression of CYP1A2, CYP2C11, CYP3A2, Bcl-2 and Bax was determined relative to that of a 9-week-old male SD reference rat, with a normal liver and without administration of haloperidol.
2.5. The serum concentration of haloperidol, AST and ALT The serum concentration of haloperidol was determined in the fatty liver group (n = 7 for 0.5 and 4 h) and the normal liver group (n = 7 for 0.5 and 4 h). The serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and 4 h after administration of haloperidol in each group were also determined. Table 1 Sequences of the primers used for RT-PCR Transcript
Sequence
CYP1A2
(F)50 -CCATGATGAGAAGCAGTGGAAA-30 (R)50 -TGGCCGTGTTGTCATTGCT-30
CYP2C11
(F)50 -CAGTCCTAGTGCTGGTGCTCACT-30 (R)50 -CTGAAGGGTGTTTCCAATGATTG-30
CYP3A2
(F)50 -GGCAAGCCTGTCACCATGA-30 (R)50 -TCGACGTTCACTCCAAATCATG-30
Bcl-2
(F)50 -TGGGATGGCTTTGTGGAACT-30 (R)50 -GAGACAGCCAGGAGAAATCAAAC-30
Bax
(F)50 -TCCCCCCGAGACGTCTTCT-30 (R)50 -CCCAGTTGAAGTTGCCATCA-30
(F) Forward primer, (R) reverse primer.
M. Hanagama et al. / Legal Medicine 10 (2008) 177–184
These concentrations were measured by a licensed laboratory (SRL, Tokyo, Japan). 2.6. Detection of apoptotic cells in the livers Three of ten samples in each group were randomly selected for morphometric analysis of apoptosis. TACSXL-Blue Label (Trevigen, Gaithersburg, MD, USA) was used for the detection of apoptosis in liver tissue, in accordance with the attached instructions. Briefly, this method can detect apoptotic cells with more sensitivity and precision than the TUNEL method. In each section, 10 microscopic fields were randomly selected under 400 magnification. The number of bluestained nuclei was counted within each field, including hepatocytes, sinusoidal lining cells and endothelial cells, and an average of 30 fields per group was calculated. Then the ratio of the average number of blue-stained nuclei in the fatty liver group to that in the normal liver group was evaluated at each time point. The values were determined relative to that at the 0-h time point. 2.7. Statistical analysis All analyses were performed using JMP version 5, Japanese edition (SAS Institute, Cary, NC, USA). The measurements were expressed as means ± SE. The difference between two groups was determined by Student’s t test or Welch’s t test. The difference among multiple groups was determined by repeated measure one-way analysis of variance. The significance of individual differences was evaluated using Tukey–Kramer’s procedure as a post hoc test. A P value of less than 0.05 was considered statistically significant. 3. Results The rats began to cramp within several minutes after administration of haloperidol. Subsequently, heart beat and breathing ceased suddenly in some of them. In the surviving rats, convulsion continued for about an hour, and then they became motionless. They recovered about half a day after administration of haloperidol. About 40% of the rats died following administration of haloperidol in both of the groups. Among the rats which died following administration of haloperidol, the average of the survival times was 26 min. There was no significant difference between the two groups in the survival rate or the survival time.
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in the normal liver continued to increase up to 4 h, and that of CYP2C11 peaked at 2 h. On the other hand, the expressions of CYP1A2, CYP2C11 and CYP3A2 showed no significant change after administration of haloperidol. In the normal liver, the expression level of CYP1A2 and CYP3A2 at 4 h was 2.7 and 1.7 times higher than that at 0 h (2.91 ± 0.34 versus 1.08 ± 0.05 and 1.71 ± 0.16 versus 0.98 ± 0.07; Fig. 1a and c) respectively, with significant difference (P < 0.0001 for both). Moreover, in the normal liver, there was significant difference between the expression level of CYP2C11 at 2 and 0 h (1.66 ± 0.24 versus 0.94 ± 0.16, P < 0.0001; Fig. 1b). 3.2. Serum concentration of haloperidol The serum concentrations of haloperidol of the fatty liver rats and the normal liver rats are shown in Table 2. At 0.5 h after administration, the concentration of haloperidol was higher in the fatty liver rats than in the normal liver rats, but the difference was not significant (1727.0 ± 129.8 versus 1387.4 ± 98.0 ng/ml, P = 0.077 by Student’s t test). The concentration of serum haloperidol at 4-h time point was significantly higher in the fatty liver rats than in the normal liver rats (958.9 ± 188.6 versus 206.3 ± 32.7 ng/ml, P < 0.0001 by Tukey–Kramer’s procedure). To evaluate the metabolic rate of haloperidol in each group, we calculated the percent decrease in the average value at 4 h compared to that at 0.5 h, which was lower in the fatty liver group than in the normal liver group (44% versus 85%). 3.3. Serum concentration of AST and ALT The serum concentrations of AST and ALT of the fatty liver rats and the normal liver rats are shown in Table 3. Before administration of haloperidol, the concentrations of both AST and ALT in the fatty liver rats were significantly higher than those in the normal liver (649.3 ± 139.7 versus 90.8 ± 7.5 IU/l; P = 0.0407 by Welch’s t test and 78.3 ± 5.4 versus 41.3 ± 2.1 IU/l; P = 0.0015 by Student’s t test). Four hours after administration of haloperidol, the serum concentrations of AST and ALT tended to increase in the fatty liver group (829.4 ± 85.9 and 166.4 ± 30.4 IU/l), but the difference was significant only in the concentrations of ALT (P = 0.0014 by Tukey–Kramer’s procedure). On the other hand, there was no significant change in the serum concentrations of AST and ALT in the normal liver group. 3.4. mRNA expression of Bcl-2 and Bax
3.1. mRNA expression of CYP1A2, CYP2C11 and CYP3A2 The expression of CYP1A2, CYP2C11 and CYP3A2 were all significantly lower in the fatty liver than in the normal liver through the experiment (P < 0.0001 by Tukey– Kramer’s procedure; Fig. 1a–c). Following administration of haloperidol, the expression of CYP1A2 and CYP3A2
Without administration of haloperidol, the mRNA expression of Bcl-2 in the fatty liver was 2.6 times higher than that in the normal liver, with significant difference (2.71 ± 0.35 versus 1.05 ± 0.07, P < 0.0001 by Tukey–Kramer’s procedure; Fig. 2a). Following administration of haloperidol, the expression of Bcl-2 in the fatty liver gradually
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M. Hanagama et al. / Legal Medicine 10 (2008) 177–184 fold fold
Normal liver rat
Fatty liver rat
Normal liver rat
2
Fatty liver rat
4
mRNA expression of CYP2C11
mRNA expression of CYP1A2
a *
3
2
1
†
†
†
†
†
b
*
1
†
0
†
†
†
†
0 0
0.5
1
2
4
h
0
0.5
1
2
4
h
fold 2
Normal liver rat
Fatty liver rat
mRNA expression of CYP3A2
c
*
1
†
†
†
†
†
0 0
0.5
1
2
4
h
Fig. 1. Time courses of mRNA expression of CYP1A2 (a), CYP2C11 (b) and CYP3A2 (c) in normal liver and fatty liver. Values are means ± SE. Without administration of haloperidol, the expression levels of CYP1A2, CYP2C11 and CYP3A2 were all lower in fatty liver than in normal liver. After administration of haloperidol, the expressions of CYP1A2, CYP2C11 and CYP3A2 were all increased significantly in normal liver but not increased in fatty liver. *Significantly different from 0 h in normal liver (P < 0.01). Significantly different from normal liver at the same time point (P < 0.01).
Table 2 Serum concentrations (ng/ml) of haloperidol in each group
Normal liver (n = 7 each) Fatty liver (n = 7 each)
0.5 h
4h
Percent decreasea
1387.4 ± 98.0 1727.0 ± 129.8
206.3 ± 32.7 958.9 ± 188.6*
85 44
Values are means ± SE. a Percent decrease in the average value at 4 h compared to that at 0.5 h in the same group. * Significantly different from normal liver at the same time point (P < 0.01).
decreased. The value at 4 h was significantly lower than that at 0 h (1.10 ± 0.09, P < 0.0001; Fig. 2a), whereas the expression of Bcl-2 showed no apparent change in the normal liver. The mRNA expression of Bax was higher in the fatty liver than in the normal liver before administration of haloperidol, without significant difference (1.14 ± 0.13 versus 0.84 ± 0.09, P = 0.0569; Fig. 2b). After administration of
haloperidol, the expression of Bax tended to increase slightly in both the fatty liver and the normal liver, but there was no significant difference. 3.5. Detection of apoptotic cells in liver tissue The number of blue-stained nuclei of liver tissues including hepatocytes, sinusoidal lining cells and endothelial cells increased in the fatty liver following administration of haloperidol, in comparison with those in pretreatment. On the other hand, the blue-stained nuclei were sparsely detected in normal liver without administration of haloperidol, but the number did not increase after administration of haloperidol (Fig. 3a–c). On morphometric analysis, the ratios of the number of blue-stained nuclei in the fatty liver tissue to that in the normal liver tissue were approximately 3.7–5 times higher after administration of haloperidol compared with the ratio in the groups without the administration of haloperidol, but without significant difference (Fig. 3d).
M. Hanagama et al. / Legal Medicine 10 (2008) 177–184 Table 3 Serum concentrations (IU/l) of AST and ALT in each group
4. Discussion
0h
4h
Normal liver AST ALT
(n = 4) 90.8 ± 7.5 41.3 ± 2.1
(n = 5) 101.2 ± 13.0 51.4 ± 4.6
Fatty liver AST ALT
(n = 4) 649.3 ± 139.7* 78.3 ± 5.4*
(n = 5) 829.4 ± 85.9* 166.4 ± 30.4*
Values are means ± SE. AST, aspartate aminotransferase; ALT, alanine aminotransferase. * Significantly different from normal liver at the same time point (P < 0.05). Significantly different from 0 h in fatty liver (P < 0.01).
fold 4 Normal liver rat
mRNA expression of Bcl-2
a
Fatty liver rat
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3
† 2
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0 0
0.5
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h
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fold
mRNA expression of Bax
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Fatty liver rat
b
1
0 0
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Fig. 2. Time courses of mRNA expression of Bcl-2 (a) and Bax (b) in normal liver and fatty liver. Values are means ± SE. Without administration of haloperidol, the expression of Bcl-2 in fatty liver was significantly higher than in normal liver. Following administration of haloperidol, the expression of Bcl-2 significantly decreased in fatty liver, whereas no apparent change was seen in normal liver. The expression of Bax slightly increased both in normal liver and in fatty liver after administration of haloperidol, but without significant difference. *Significantly different from 0 h in fatty liver (P < 0.01). Significantly different from normal liver at the same time point (P < 0.01).
In this study, the mRNA expressions of CYP1A2, CYP2C11 and CYP3A2 were all downregulated in fatty liver, which agreed with previous reports [18,24–27]. Gene expressions of CYPs are regulated by extracellular and intracellular signals via nuclear receptors [28]. Several reports have suggested that alterations in the expression of nuclear receptors contribute to CYP dysregulation in fatty liver [18,27–30]. Additionally, decreased serum testosterone may cause downregulation of CYP2C11 and CYP3A2 in fatty liver rats with choline-deficient diet, because these CYPs are induced by testosterone [20,24,31]. Our experiment also showed impaired responses of the mRNA expressions of CYPs to haloperidol in the fatty liver. Since drugs interact with nuclear receptors to induce CYP enzymes [28], it is likely that alterations in the expressions of nuclear receptors caused different responses of the mRNA expressions of CYPs to haloperidol between the normal liver and the fatty liver. In addition to suppressed CYP expression, this study showed significant decrease in metabolism of haloperidol in fatty liver rats. Rat CYP3A2 catalyzes N-dealkylation of haloperidol, which is a major pathway of haloperidol metabolism in the liver [32]. It is thus likely that the decreased expression of CYP3A2 in the fatty liver mainly affected the metabolism of haloperidol. On the other hand, the contributions of CYP1A2 and CYP2C11 to the metabolism of haloperidol are reported to be less than that of CYP3A2 [32]; however, the induction of CYP1A2 and CYP2C11 by administration of haloperidol in the normal liver suggested that these enzymes might partially metabolize haloperidol. Besides the suppressed expressions of CYPs, accumulated lipid in hepatocytes may directly inhibit activities of CYP enzymes [27]. Therefore, these results strongly suggest that metabolism of various drugs including haloperidol is impaired in fatty liver. Actually, psychiatric patients are often treated with multiple drugs such as antipsychotics, antidepressants and tranquilizers [33], which may cause enhanced adverse effects in patients with fatty liver, leading to serious results such as coma and death in the worst case. It is generally accepted that fatty liver occasionally suffers oxidative stress, dysfunction of mitochondria, dysregulation of cytokines and/or apoptosis, leading to hepatocellular and tissue injuries [34–42]. It has been reported that death receptors such as TNF-R1 and Fas, which are upstream of apoptosis pathways including Bcl2 and Bax, are upregulated in fatty liver [38–42]. In our study, the mRNA expression of Bcl-2 was significantly higher in the fatty liver than in the normal liver without administration of haloperidol. Moreover, both AST and ALT increased in the fatty liver rats. A previous report has also shown that the expression of Bcl-2 is increased in proportion to the degree of liver injury in fatty liver [41]. Therefore, it is likely that upregulation of Bcl-2 in fatty liver is an adaptive response to injurious stimuli including induction of apoptosis.
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The ratio of number of blue-stained nuclei
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fold
6
4
2
0
0.5
1
2
4
h
Fig. 3. Fatty and normal liver tissues stained by TACS-XL-Blue Label and the morphometric analysis following administration of haloperidol. Large numbers of blue-stained nuclei of hepatocytes (black arrowhead) are seen in fatty liver tissues at 0.5 h (a) and 4 h (b) after administration of haloperidol, compared with that in normal liver tissue at 4 h after administration of haloperidol (c) (200 each). On the morphometric analysis (d), the ratio of the number of blue-stained nuclei between the fatty liver tissue and the normal liver tissue increases after administration of haloperidol compared with that without haloperidol.
Haloperidol can induce apoptosis or oxidative stress, leading to cytotoxicity [43–47]. In this study, the expression of Bax slightly increased in both the normal liver and the fatty liver after administration of haloperidol, whereas the expression of Bcl-2 significantly decreased only in the fatty liver. These results suggested that haloperidol stimulated apoptosis pathways in the liver. In particular, the decrease of Bcl-2 expression in the fatty liver following administration of haloperidol suggested that haloperidol attenuated the adaptive response to the previously-existing apoptotic stimuli. In addition, prolonged high concentration of serum haloperidol in the fatty liver rats might contribute to increasing apoptotic stimuli. The morphometric analysis also showed that the fatty liver tissues were more susceptible to induction of apoptosis by haloperidol than the normal liver tissues. However, the time course of the ratio of the average number of apoptotic cells between fatty liver and normal liver in morphometric analysis was incompatible with the time courses in the expression of Bcl-2 and Bax. Previous reports have shown that other Bcl-2 family proteins and mitochondrial dysfunction may contribute
to haloperidol-induced apoptosis via sigma-2 receptor, which is expressed in various tissues including the liver [43–46,48], suggesting the contribution of other factors besides Bcl-2 and Bax to the induction of apoptosis shown in our experiment. On the other hand, pyridinic metabolites of haloperidol produced by CYP enzymes and druginduced CYPs themselves can cause injurious oxidative stress [49,50]. However, in spite of the higher expressions of CYPs in the normal liver than in the fatty liver, AST and ALT in the normal liver rats did not increase after administration of haloperidol in our study. It is thus likely that hepatic cytotoxicity of haloperidol might be induced by apoptosis rather than oxidative stress. Hepatotoxicity of haloperidol has been reported as a rare side effect [51,52]. However, our results suggest that haloperidol causes strong apoptotic stimuli and may lead to progression of cellular and tissue injury in fatty liver. In conclusion, our study demonstrated that gene expression of CYPs decreased, and that the response of CYPs to haloperidol was reduced in the fatty liver. Moreover, severe fatty liver may reinforce cytotoxic effects of haloperidol.
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Thus, it is possible that individuals with severe fatty liver are susceptible to adverse effects of antipsychotic drugs including haloperidol, leading to life-threatening events. It is important to further investigate the adverse effects of antipsychotic drugs on fatty liver to elucidate the side effects of these drugs. Accordingly, it should be noted by clinicians that excess dose of antipsychotic drugs may be more harmful in patients with fatty liver than in those with normal liver. Acknowledgement The authors thank Dr. Myles O’Brien (Mie Prefectural College of Nursing, Tsu, Mie, Japan) for revising the English used in this article. References [1] Hugenholtz GW, Heerdink ER, Stolker JJ, Meijer WE, Egberts AC, Nolen WA. Haloperidol dose when used as active comparator in randomized controlled trials with atypical antipsychotics in schizophrenia: comparison with officially recommended doses. J Clin Psychiatry 2006;67:897–903. [2] Usuki E, Van der Schyf CJ, Castagnoli Jr N. Metabolism of haloperidol and its tetrahydropyridine dehydration product HPTP. Drug Metab Rev 1998;30:809–26. [3] Rosh A, Sampson BA, Hirsch CS. Schizophrenia as a cause of death. J Forensic Sci 2003;48:164–7. [4] Hennessy S, Bilker WB, Knauss JS, Margolis DJ, Kimmel SE, Reynolds RF, et al. Cardiac arrest and ventricular arrhythmia in patients taking antipsychotic drugs: cohort study using administrative data. BMJ 2002;325:1070–4. [5] Straus SM, Bleumink GS, Dieleman JP, van der Lei J, ’t Jong GW, Kingma JH, et al. Antipsychotics and the risk of sudden cardiac death. Arch Intern Med 2004;164:1293–7. [6] Reilly JG, Ayis SA, Ferrier IN, Jones SJ, Thomas SH. QTc-interval abnormalities and psychotropic drug therapy in psychiatric patients. Lancet 2000;355:1048–52. [7] McEvoy JP, Meyer JM, Goff DC, Nasrallah HA, Davis SM, Sullivan L, et al. Prevalence of the metabolic syndrome in patients with schizophrenia: baseline results from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) schizophrenia trial and comparison with national estimates from NHANES III. Schizophr Res 2005;80:19–32. [8] Ha¨gg S, Lindblom Y, Mjorndal T, Adolfsson R. High prevalence of the metabolic syndrome among a Swedish cohort of patients with schizophrenia. Int Clin Psychopharmacol 2006;21:93–8. [9] Kim HJ, Kim HJ, Lee KE, Kim DJ, Kim SK, Ahn CW, et al. Metabolic significance of nonalcoholic fatty liver disease in nonobese, nondiabetic adults. Arch Intern Med 2004;164:2169–75. [10] Adams LA, Angulo P. Recent concepts in non-alcoholic fatty liver disease. Diabet Med 2005;22:1129–33. [11] Machado M, Cortez-Pinto H. Non-alcoholic steatohepatitis and metabolic syndrome. Curr Opin Clin Nutr Metab Care 2006;9:637–42. [12] Durlacher SH, Meier JR, Fisher RS, Lovitt Jr WV. Sudden death due to pulmonary fat embolism in chronic alcoholics with fatty livers. J Forensic Sci 1959;4:215–28. [13] Randall B. Fatty liver and sudden death. Hum Pathol 1980;11:147–53. [14] Jones MB. Pulmonary fat emboli associated with acute fatty liver of pregnancy. Am J Gastroenterol 1993;88:791–2. [15] Cheymol G. Effects of obesity on pharmacokinetics implications for drug therapy. Clin Pharmacokinet 2000;39:215–31.
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Gene expression on liver toxicity induced by administration of haloperidol in rats with severe fatty liver Masakazu Hanagama, Hiromasa Inoue *, Munechika Kamiya, Kotaro Shinone, Masayuki Nata Department of Forensic Medicine and Sciences, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu City, Mie 514-8507, Japan Received 21 August 2007; received in revised form 14 December 2007; accepted 26 December 2007 Available online 14 February 2008
Abstract Sudden deaths are often encountered in schizophrenic patients prescribed with antipsychotic drugs, and fatty liver may be more prevalent among patients with schizophrenia. The aim of this study is to investigate the adverse effects of antipsychotic drugs on fatty liver. We administered haloperidol intraperitoneally to fatty liver rats and examined the mRNA expression in the liver. Basic expressions of cytochrome P450 (CYP)1A2, CYP2C11 and CYP3A2 decreased, and response of these CYPs to haloperidol was reduced in the fatty liver. Metabolism of haloperidol was also suppressed in the fatty liver rats. Moreover, hepatic injury by administration of haloperidol was shown pathohistologically and molecular-biologically in severe fatty liver. These results suggest that fatty liver increases susceptibility to adverse effects of haloperidol, possibly leading to life-threatening events. It should be noted by clinicians that excessive dose of antipsychotic drugs may be more harmful in patients with fatty liver. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Schizophrenia; Sudden death; Fatty liver; Haloperidol; Cytochrome P450; Apoptosis
1. Introduction Antipsychotic drugs have been widely used in treatment of schizophrenia [1,2]. In forensic practices, sudden deaths are often encountered in schizophrenic patients, in which it is often difficult to decide the precise causes of death after performing complete autopsy [3]. One of the mechanisms of death might be the occurrence of ventricular arrhythmia and QT prolongation associated with antipsychotic substances, resulting in sudden cardiac death [4–6]; however, it has hardly been investigated whether the effects of these drugs on the other organs contribute to death. Schizophrenic patients are reported to have higher incidence of obesity, diabetes mellitus and dyslipidemia than in the general population [7,8]. These metabolic disorders are closely associated with fatty liver, indicating that fatty liver
*
Corresponding author. Tel./fax: +81 59 231 5014. E-mail address: [email protected] (H. Inoue).
may well be more prevalent among patients with schizophrenia than among the general population, where its occurrence is estimated at 10–34% [9–11]. In forensic medicine, there have been many reports of sudden death cases associated with fatty liver [12–14]. Moreover, it is suggested that drug metabolism may be impaired in patients with fatty liver [15]. However, there have been no reports of investigation into the relationship between the effect of antipsychotic drugs on fatty liver and death. Therefore, this study aimed to investigate the adverse effects of antipsychotic drugs on fatty and normal liver. Haloperidol is one of the most widely used antipsychotic drugs for the treatment of schizophrenia [1]. We administered it to normal rats and fatty liver rats, and from the point of view of drug metabolism and tissue injury, we examined mRNA expression in the liver of cytochrome P450 (CYP)1A2, CYP2C11 and CYP3A2, which are major metabolic enzymes of various drugs in the liver [16–20], and Bcl-2 and Bax, which are anti- and pro-apoptotic proteins [21,22].
1344-6223/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2007.12.006
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2. Materials and methods
2.3. RNA extraction and complementary cDNA synthesis
The experiments were reviewed by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Mie University and carried out under the control of Guidelines for Animal Experiments in the Faculty of Medicine, Mie University and the Law (No. 105) and Notification (No. 6) of the Government of Japan. Moreover, this study followed the ‘‘Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
mRNA was extracted from the liver using QuickGene RNA tissue kit SII (FUJIFILM, Tokyo, Japan) according to the manufacturer’s instructions. Then, this mRNA was utilized as a template, and double-stranded complementary DNA (cDNA) was synthesized by using a first-strand cDNA synthesis kit (ReverTra Ace-a-, Toyobo, Osaka, Japan), according to the attached instructions.
2.4. Polymerase chain reaction quantification of mRNA expression
2.1. Preliminary experiment To induce fatty liver, 3-week-old male Sprague–Dawley (SD) rats (CLEA Japan, Inc., Tokyo, Japan) were reared for 6 weeks with only a special laboratory feed [23]. All the rats were housed in a temperature-controlled room on a 12:12 light–dark cycle. The fatty liver rats (100–170 g) were administered various doses of haloperidol (SerenaceÒ, Dainippon Sumitomo Pharma, Osaka, Japan). When the rats were injected with 40 mg/kg haloperidol intraperitoneally, almost half of them died about 30-min after administration, but the others survived. Accordingly, we adopted 40 mg/kg of haloperidol as an intoxicating dose for rats in this study. 2.2. Animal treatment The experiment was performed to compare two groups: (I) rats with fatty liver as the fatty liver group (50–170 g) and (II) 9- to 12-week-old male SD rats without fatty liver as the normal liver group (210–390 g). Normal liver rats and fatty liver rats were injected with 40 mg/kg haloperidol intraperitoneally. Because almost half of those rats died about 30-min after administration of haloperidol in the preliminary study, survival time for them was regarded as 0.5 h. The surviving rats were killed in a short time in a vacuum chamber at 1, 2 and 4 h after administration of haloperidol. The liver was removed immediately after death, and a portion of it was soaked in RNAlater (Applied Biosystems, Foster City, CA, USA) for one night and then stored at 80 °C for later analysis of mRNA expression. In addition, the other portion of the liver was fixed in 4% formaldehyde, embedded in paraffin, and then 4-lm thick sections were cut for the pathohistological detection of apoptotic cells. We randomly chose the liver samples to analyze from both the fatty liver group (n = 10 for 0.5, 1, 2 and 4 h) and the normal liver group (n = 10 for 0.5, 1, 2 and 4 h). As two pretreatment controls, 9-week-old SD rats without fatty liver (n = 10) and with fatty liver (n = 10), but without administration of haloperidol, were killed and then treated using the same methods.
Primer sequences for cytochrome P450 (CYP)1A2, CYP2C11, CYP3A2, Bcl-2 and Bax cDNA, which were designed using Primer Express version 1.5 (Applied Biosystems, Foster City, CA, USA), are presented in Table 1. The mRNA levels of CYP1A2, CYP2C11, CYP3A2, Bcl-2, Bax and b-actin (an internal reference cDNA) were quantified using a fluorescence detection system (ABI PRISM 7700 sequence detection system, Applied Biosystems), in accordance with the attached instructions. The PCR mixture consisted of 0.5 lM of each primer (CYP1A2, CYP2C11, CYP3A2 Bcl-2 or Bax) and 5 ll of Power SYBR Green Realtime PCR Master Mix (Applied Biosystems) in a final volume of 10 ll. The expression of CYP1A2, CYP2C11, CYP3A2, Bcl-2 and Bax was determined relative to that of a 9-week-old male SD reference rat, with a normal liver and without administration of haloperidol.
2.5. The serum concentration of haloperidol, AST and ALT The serum concentration of haloperidol was determined in the fatty liver group (n = 7 for 0.5 and 4 h) and the normal liver group (n = 7 for 0.5 and 4 h). The serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and 4 h after administration of haloperidol in each group were also determined. Table 1 Sequences of the primers used for RT-PCR Transcript
Sequence
CYP1A2
(F)50 -CCATGATGAGAAGCAGTGGAAA-30 (R)50 -TGGCCGTGTTGTCATTGCT-30
CYP2C11
(F)50 -CAGTCCTAGTGCTGGTGCTCACT-30 (R)50 -CTGAAGGGTGTTTCCAATGATTG-30
CYP3A2
(F)50 -GGCAAGCCTGTCACCATGA-30 (R)50 -TCGACGTTCACTCCAAATCATG-30
Bcl-2
(F)50 -TGGGATGGCTTTGTGGAACT-30 (R)50 -GAGACAGCCAGGAGAAATCAAAC-30
Bax
(F)50 -TCCCCCCGAGACGTCTTCT-30 (R)50 -CCCAGTTGAAGTTGCCATCA-30
(F) Forward primer, (R) reverse primer.
M. Hanagama et al. / Legal Medicine 10 (2008) 177–184
These concentrations were measured by a licensed laboratory (SRL, Tokyo, Japan). 2.6. Detection of apoptotic cells in the livers Three of ten samples in each group were randomly selected for morphometric analysis of apoptosis. TACSXL-Blue Label (Trevigen, Gaithersburg, MD, USA) was used for the detection of apoptosis in liver tissue, in accordance with the attached instructions. Briefly, this method can detect apoptotic cells with more sensitivity and precision than the TUNEL method. In each section, 10 microscopic fields were randomly selected under 400 magnification. The number of bluestained nuclei was counted within each field, including hepatocytes, sinusoidal lining cells and endothelial cells, and an average of 30 fields per group was calculated. Then the ratio of the average number of blue-stained nuclei in the fatty liver group to that in the normal liver group was evaluated at each time point. The values were determined relative to that at the 0-h time point. 2.7. Statistical analysis All analyses were performed using JMP version 5, Japanese edition (SAS Institute, Cary, NC, USA). The measurements were expressed as means ± SE. The difference between two groups was determined by Student’s t test or Welch’s t test. The difference among multiple groups was determined by repeated measure one-way analysis of variance. The significance of individual differences was evaluated using Tukey–Kramer’s procedure as a post hoc test. A P value of less than 0.05 was considered statistically significant. 3. Results The rats began to cramp within several minutes after administration of haloperidol. Subsequently, heart beat and breathing ceased suddenly in some of them. In the surviving rats, convulsion continued for about an hour, and then they became motionless. They recovered about half a day after administration of haloperidol. About 40% of the rats died following administration of haloperidol in both of the groups. Among the rats which died following administration of haloperidol, the average of the survival times was 26 min. There was no significant difference between the two groups in the survival rate or the survival time.
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in the normal liver continued to increase up to 4 h, and that of CYP2C11 peaked at 2 h. On the other hand, the expressions of CYP1A2, CYP2C11 and CYP3A2 showed no significant change after administration of haloperidol. In the normal liver, the expression level of CYP1A2 and CYP3A2 at 4 h was 2.7 and 1.7 times higher than that at 0 h (2.91 ± 0.34 versus 1.08 ± 0.05 and 1.71 ± 0.16 versus 0.98 ± 0.07; Fig. 1a and c) respectively, with significant difference (P < 0.0001 for both). Moreover, in the normal liver, there was significant difference between the expression level of CYP2C11 at 2 and 0 h (1.66 ± 0.24 versus 0.94 ± 0.16, P < 0.0001; Fig. 1b). 3.2. Serum concentration of haloperidol The serum concentrations of haloperidol of the fatty liver rats and the normal liver rats are shown in Table 2. At 0.5 h after administration, the concentration of haloperidol was higher in the fatty liver rats than in the normal liver rats, but the difference was not significant (1727.0 ± 129.8 versus 1387.4 ± 98.0 ng/ml, P = 0.077 by Student’s t test). The concentration of serum haloperidol at 4-h time point was significantly higher in the fatty liver rats than in the normal liver rats (958.9 ± 188.6 versus 206.3 ± 32.7 ng/ml, P < 0.0001 by Tukey–Kramer’s procedure). To evaluate the metabolic rate of haloperidol in each group, we calculated the percent decrease in the average value at 4 h compared to that at 0.5 h, which was lower in the fatty liver group than in the normal liver group (44% versus 85%). 3.3. Serum concentration of AST and ALT The serum concentrations of AST and ALT of the fatty liver rats and the normal liver rats are shown in Table 3. Before administration of haloperidol, the concentrations of both AST and ALT in the fatty liver rats were significantly higher than those in the normal liver (649.3 ± 139.7 versus 90.8 ± 7.5 IU/l; P = 0.0407 by Welch’s t test and 78.3 ± 5.4 versus 41.3 ± 2.1 IU/l; P = 0.0015 by Student’s t test). Four hours after administration of haloperidol, the serum concentrations of AST and ALT tended to increase in the fatty liver group (829.4 ± 85.9 and 166.4 ± 30.4 IU/l), but the difference was significant only in the concentrations of ALT (P = 0.0014 by Tukey–Kramer’s procedure). On the other hand, there was no significant change in the serum concentrations of AST and ALT in the normal liver group. 3.4. mRNA expression of Bcl-2 and Bax
3.1. mRNA expression of CYP1A2, CYP2C11 and CYP3A2 The expression of CYP1A2, CYP2C11 and CYP3A2 were all significantly lower in the fatty liver than in the normal liver through the experiment (P < 0.0001 by Tukey– Kramer’s procedure; Fig. 1a–c). Following administration of haloperidol, the expression of CYP1A2 and CYP3A2
Without administration of haloperidol, the mRNA expression of Bcl-2 in the fatty liver was 2.6 times higher than that in the normal liver, with significant difference (2.71 ± 0.35 versus 1.05 ± 0.07, P < 0.0001 by Tukey–Kramer’s procedure; Fig. 2a). Following administration of haloperidol, the expression of Bcl-2 in the fatty liver gradually
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M. Hanagama et al. / Legal Medicine 10 (2008) 177–184 fold fold
Normal liver rat
Fatty liver rat
Normal liver rat
2
Fatty liver rat
4
mRNA expression of CYP2C11
mRNA expression of CYP1A2
a *
3
2
1
†
†
†
†
†
b
*
1
†
0
†
†
†
†
0 0
0.5
1
2
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h
0
0.5
1
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h
fold 2
Normal liver rat
Fatty liver rat
mRNA expression of CYP3A2
c
*
1
†
†
†
†
†
0 0
0.5
1
2
4
h
Fig. 1. Time courses of mRNA expression of CYP1A2 (a), CYP2C11 (b) and CYP3A2 (c) in normal liver and fatty liver. Values are means ± SE. Without administration of haloperidol, the expression levels of CYP1A2, CYP2C11 and CYP3A2 were all lower in fatty liver than in normal liver. After administration of haloperidol, the expressions of CYP1A2, CYP2C11 and CYP3A2 were all increased significantly in normal liver but not increased in fatty liver. *Significantly different from 0 h in normal liver (P < 0.01). Significantly different from normal liver at the same time point (P < 0.01).
Table 2 Serum concentrations (ng/ml) of haloperidol in each group
Normal liver (n = 7 each) Fatty liver (n = 7 each)
0.5 h
4h
Percent decreasea
1387.4 ± 98.0 1727.0 ± 129.8
206.3 ± 32.7 958.9 ± 188.6*
85 44
Values are means ± SE. a Percent decrease in the average value at 4 h compared to that at 0.5 h in the same group. * Significantly different from normal liver at the same time point (P < 0.01).
decreased. The value at 4 h was significantly lower than that at 0 h (1.10 ± 0.09, P < 0.0001; Fig. 2a), whereas the expression of Bcl-2 showed no apparent change in the normal liver. The mRNA expression of Bax was higher in the fatty liver than in the normal liver before administration of haloperidol, without significant difference (1.14 ± 0.13 versus 0.84 ± 0.09, P = 0.0569; Fig. 2b). After administration of
haloperidol, the expression of Bax tended to increase slightly in both the fatty liver and the normal liver, but there was no significant difference. 3.5. Detection of apoptotic cells in liver tissue The number of blue-stained nuclei of liver tissues including hepatocytes, sinusoidal lining cells and endothelial cells increased in the fatty liver following administration of haloperidol, in comparison with those in pretreatment. On the other hand, the blue-stained nuclei were sparsely detected in normal liver without administration of haloperidol, but the number did not increase after administration of haloperidol (Fig. 3a–c). On morphometric analysis, the ratios of the number of blue-stained nuclei in the fatty liver tissue to that in the normal liver tissue were approximately 3.7–5 times higher after administration of haloperidol compared with the ratio in the groups without the administration of haloperidol, but without significant difference (Fig. 3d).
M. Hanagama et al. / Legal Medicine 10 (2008) 177–184 Table 3 Serum concentrations (IU/l) of AST and ALT in each group
4. Discussion
0h
4h
Normal liver AST ALT
(n = 4) 90.8 ± 7.5 41.3 ± 2.1
(n = 5) 101.2 ± 13.0 51.4 ± 4.6
Fatty liver AST ALT
(n = 4) 649.3 ± 139.7* 78.3 ± 5.4*
(n = 5) 829.4 ± 85.9* 166.4 ± 30.4*
Values are means ± SE. AST, aspartate aminotransferase; ALT, alanine aminotransferase. * Significantly different from normal liver at the same time point (P < 0.05). Significantly different from 0 h in fatty liver (P < 0.01).
fold 4 Normal liver rat
mRNA expression of Bcl-2
a
Fatty liver rat
†
†
3
† 2
* 1
0 0
0.5
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h
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h
fold
mRNA expression of Bax
2
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Fatty liver rat
b
1
0 0
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Fig. 2. Time courses of mRNA expression of Bcl-2 (a) and Bax (b) in normal liver and fatty liver. Values are means ± SE. Without administration of haloperidol, the expression of Bcl-2 in fatty liver was significantly higher than in normal liver. Following administration of haloperidol, the expression of Bcl-2 significantly decreased in fatty liver, whereas no apparent change was seen in normal liver. The expression of Bax slightly increased both in normal liver and in fatty liver after administration of haloperidol, but without significant difference. *Significantly different from 0 h in fatty liver (P < 0.01). Significantly different from normal liver at the same time point (P < 0.01).
In this study, the mRNA expressions of CYP1A2, CYP2C11 and CYP3A2 were all downregulated in fatty liver, which agreed with previous reports [18,24–27]. Gene expressions of CYPs are regulated by extracellular and intracellular signals via nuclear receptors [28]. Several reports have suggested that alterations in the expression of nuclear receptors contribute to CYP dysregulation in fatty liver [18,27–30]. Additionally, decreased serum testosterone may cause downregulation of CYP2C11 and CYP3A2 in fatty liver rats with choline-deficient diet, because these CYPs are induced by testosterone [20,24,31]. Our experiment also showed impaired responses of the mRNA expressions of CYPs to haloperidol in the fatty liver. Since drugs interact with nuclear receptors to induce CYP enzymes [28], it is likely that alterations in the expressions of nuclear receptors caused different responses of the mRNA expressions of CYPs to haloperidol between the normal liver and the fatty liver. In addition to suppressed CYP expression, this study showed significant decrease in metabolism of haloperidol in fatty liver rats. Rat CYP3A2 catalyzes N-dealkylation of haloperidol, which is a major pathway of haloperidol metabolism in the liver [32]. It is thus likely that the decreased expression of CYP3A2 in the fatty liver mainly affected the metabolism of haloperidol. On the other hand, the contributions of CYP1A2 and CYP2C11 to the metabolism of haloperidol are reported to be less than that of CYP3A2 [32]; however, the induction of CYP1A2 and CYP2C11 by administration of haloperidol in the normal liver suggested that these enzymes might partially metabolize haloperidol. Besides the suppressed expressions of CYPs, accumulated lipid in hepatocytes may directly inhibit activities of CYP enzymes [27]. Therefore, these results strongly suggest that metabolism of various drugs including haloperidol is impaired in fatty liver. Actually, psychiatric patients are often treated with multiple drugs such as antipsychotics, antidepressants and tranquilizers [33], which may cause enhanced adverse effects in patients with fatty liver, leading to serious results such as coma and death in the worst case. It is generally accepted that fatty liver occasionally suffers oxidative stress, dysfunction of mitochondria, dysregulation of cytokines and/or apoptosis, leading to hepatocellular and tissue injuries [34–42]. It has been reported that death receptors such as TNF-R1 and Fas, which are upstream of apoptosis pathways including Bcl2 and Bax, are upregulated in fatty liver [38–42]. In our study, the mRNA expression of Bcl-2 was significantly higher in the fatty liver than in the normal liver without administration of haloperidol. Moreover, both AST and ALT increased in the fatty liver rats. A previous report has also shown that the expression of Bcl-2 is increased in proportion to the degree of liver injury in fatty liver [41]. Therefore, it is likely that upregulation of Bcl-2 in fatty liver is an adaptive response to injurious stimuli including induction of apoptosis.
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The ratio of number of blue-stained nuclei
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6
4
2
0
0.5
1
2
4
h
Fig. 3. Fatty and normal liver tissues stained by TACS-XL-Blue Label and the morphometric analysis following administration of haloperidol. Large numbers of blue-stained nuclei of hepatocytes (black arrowhead) are seen in fatty liver tissues at 0.5 h (a) and 4 h (b) after administration of haloperidol, compared with that in normal liver tissue at 4 h after administration of haloperidol (c) (200 each). On the morphometric analysis (d), the ratio of the number of blue-stained nuclei between the fatty liver tissue and the normal liver tissue increases after administration of haloperidol compared with that without haloperidol.
Haloperidol can induce apoptosis or oxidative stress, leading to cytotoxicity [43–47]. In this study, the expression of Bax slightly increased in both the normal liver and the fatty liver after administration of haloperidol, whereas the expression of Bcl-2 significantly decreased only in the fatty liver. These results suggested that haloperidol stimulated apoptosis pathways in the liver. In particular, the decrease of Bcl-2 expression in the fatty liver following administration of haloperidol suggested that haloperidol attenuated the adaptive response to the previously-existing apoptotic stimuli. In addition, prolonged high concentration of serum haloperidol in the fatty liver rats might contribute to increasing apoptotic stimuli. The morphometric analysis also showed that the fatty liver tissues were more susceptible to induction of apoptosis by haloperidol than the normal liver tissues. However, the time course of the ratio of the average number of apoptotic cells between fatty liver and normal liver in morphometric analysis was incompatible with the time courses in the expression of Bcl-2 and Bax. Previous reports have shown that other Bcl-2 family proteins and mitochondrial dysfunction may contribute
to haloperidol-induced apoptosis via sigma-2 receptor, which is expressed in various tissues including the liver [43–46,48], suggesting the contribution of other factors besides Bcl-2 and Bax to the induction of apoptosis shown in our experiment. On the other hand, pyridinic metabolites of haloperidol produced by CYP enzymes and druginduced CYPs themselves can cause injurious oxidative stress [49,50]. However, in spite of the higher expressions of CYPs in the normal liver than in the fatty liver, AST and ALT in the normal liver rats did not increase after administration of haloperidol in our study. It is thus likely that hepatic cytotoxicity of haloperidol might be induced by apoptosis rather than oxidative stress. Hepatotoxicity of haloperidol has been reported as a rare side effect [51,52]. However, our results suggest that haloperidol causes strong apoptotic stimuli and may lead to progression of cellular and tissue injury in fatty liver. In conclusion, our study demonstrated that gene expression of CYPs decreased, and that the response of CYPs to haloperidol was reduced in the fatty liver. Moreover, severe fatty liver may reinforce cytotoxic effects of haloperidol.
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