1521-009X/43/9/1345–1352$25.00 http://dx.doi.org/10.1124/dmd.115.064980DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1345–1352, September 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Damage to the Brain Serotonergic System Increases the Expressionof Liver Cytochrome P450

Marta Rysz, Ewa Bromek, Anna Haduch, Anna Sadakierska-Chudy, and Władysława A. Daniel

Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland

Received April 17, 2015; accepted June 9, 2015

ABSTRACT

Genes coding for cytochrome P450 are regulated by endogenoushormones such as the growth hormone, corticosteroids, thyroid,and sex hormones. Secretion of these hormones is regulated bythe respective hypothalamus–pituitary–secretory organ axes.Since the brain sends its serotonergic projections from the raphenuclei to the hypothalamus, we have assumed that damage tothese nuclei may affect the neuroendocrine regulation of cyto-chrome P450 expression in the liver. Thereby, 5,7-dihydroxytryptamine(5,7-DHT), a serotonergic neurotoxin, was injected into the dor-sal and median raphe nuclei of male Wistar rats. Ten days afterthe neurotoxin injections, the brain concentrations of neuro-transmitters, serum hormone, and cytokine levels, as well as theexpression of cytochrome P450 in the liver were measured.Injection of 5,7-DHT decreased serotonin concentration in the

brain followed by a significant rise in the levels of the growthhormone, corticosterone, and testosterone, and a drop in tri-iodothyronine concentration in the serum. No changes in in-terleukin (IL) levels (IL-2 and IL-6) were observed. Simultaneously,the activity and protein level of liver CYP1A, CYP3A1, andCYP2C11 rose (the activity of CYP2A/2B/2C6/2D was not signifi-cantly changed). Similarly, the mRNA levels of CYP1A1, CYP1A2,CYP2C11, and CYP3A1 were elevated. This is the first reportdemonstrating the effect of intracerebral administration of seroto-nergic neurotoxin on liver cytochrome P450. The obtained resultsindicate involvement of the brain serotonergic system in theneuroendocrine regulation of liver cytochrome P450 expression.The physiologic and pharmacological significance of the findings isdiscussed.

Introduction

The role of the nervous system in the regulation of cytochromeP450 (P450) expression has not yet been fully recognized. Since thesecretion of hormones regulating cytochrome P450 genes (the growthhormone, corticosteroids, thyroid hormones, sex hormones) iscontrolled by the nervous system (Müller, 1989; McMahon et al.,2001), changes in brain neurotransmission may influence cytochromeP450 expression in a neurotransmitter-dependent way (Wójcikowskiand Daniel, 2011).The following axes play a key role in the hormonal regulation of

hepatic cytochrome P450: the hypothalamic-pituitary-adrenal axis(which regulates cortisol/corticosterone level), the hypothalamic-pituitary-thyroidal axis (which controls triiodothyronine and thyroxinelevels), and the so-called hypothalamic-pituitary-hepatic axis (whichaffects growth hormone level). These three axes are controlled by thebrain nervous system, in particular by the densely and diverselyinnervated hypothalamus (Törk, 1990; Lechin et al., 2006). Thehypothalamus contains the paraventricular nucleus (which producesthe corticotropin-releasing hormone, the thyrotropin-releasing hormone,

and the growth hormone release–inhibiting hormone somatostatin) andthe arcuate nucleus (which synthesizes the growth hormone–releasinghormone), which affects the secretion of hormones from the anterior lobeof the pituitary gland, and these hormones are engaged in the regulationof P450 gene expression. CYP2C11 is the main male rat isoform in theliver, being stimulated by pulsatile growth hormone (GH) secretion. Theexpression of male rat CYP2A2, CYP2C13, CYP3A2, and CYP4A2, aswell as CYP2B1/2, CYP3A2, and CYP3A18, dominant in males, alsodepends on pulsatile GH secretion (Waxman et al., 1995; Waxman andO’Connor, 2006). Corticosterone is a positive regulator of CYP1A andCYP3A (Gibson et al., 2002; Monostory et al., 2005), whereas thyroidhormones negatively affect the expression of different P450 isoforms(Yamazoe et al., 1989; Murayama et al., 1991; Liddle et al., 1998). Ourrecent studies have provided a vast body of direct evidence that braindopaminergic (Wójcikowski et al., 2007, 2008) and noradrenergic(Bromek et al., 2013; Sadakierska-Chudy et al., 2013) systems regulatecytochrome P450 expression via a neuroendocrine mechanism.The hypothalamus is innervated by projections originating from the

serotonergic anterior raphe nuclei, mainly from the dorsal (DRN) andmedian (MRN) raphe nuclei. Serotonergic projections reach theparaventricular and arcuate nuclei (Sawchenko et al., 1983; Gruberet al., 1987; Willoughby and Blessing, 1987; Larsen et al., 1996). Theeffect of the brain serotonergic system on the regulation of pituitaryhormones secretion has not been satisfactorily explained, since bothstimulatory and inhibitory actions can be observed (Tuomisto andMännistö, 1985; Müller, 1989). A number of studies showed a positive

This work was financially supported by the Interdisciplinary PhD Studies project“Molecular sciences for medicine” (cofinanced by the European Social Fundwithin the Human Capital Operational Programme) and by statutory funds fromthe Institute of Pharmacology, Polish Academy of Sciences.

dx.doi.org/10.1124/dmd.115.064980.

ABBREVIATIONS: AhR, aryl hydrocarbon receptor; 5,7-DHT, 5,7-dihydroxytryptamine; DRN, dorsal raphe nucleus; ELISA, enzyme-linkedimmunosorbent assay; GH, growth hormone; 5-HIAA, metabolite 5-hydroxyindoleacetic acid; HPLC, high-performance liquid chromatography; IL,interleukin; MRN, median raphe nucleus; P450, cytochrome P450; PCA, p-chloroamphetamine; PCPA, p-chlorophenylalanine; PCR, polymerasechain reaction; T3, triiodothyronine; T4, thyroxine; TSH, thyroid-stimulating hormone.

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effect of brain serotonin on the secretion of adrenocorticotropichormone (Jørgensen, 2007). The stimulating effect of the brainserotonergic system on growth hormone secretion can be initiated byeither growth hormone–releasing hormone release from the arcuatenucleus (Vijayan et al., 1978; Murakami et al., 1986; Willoughbyet al., 1987) or the suppression of somatostatin release from theparaventricular nucleus (Mota et al., 1995; Valverde et al., 2000). Amajority of data indicate a complex mechanism of the serotonin-regulated thyroid-stimulating hormone (TSH) secretion which mayoccur at the level of the hypothalamus (inhibition of thyrotropin-releasing hormone release or somatostatin secretion) or pituitary gland(stimulation of TSH secretion) (Abrahamson et al., 1987; Toivonenet al., 1990; Masalova and Sapronov, 2009).Our preliminary studies into the general damage of the serotonergic

system (central and peripheral) indicate involvement of this system inthe regulation of cytochrome P450. Intraperitoneal administration ofthe neurotoxin p-chloroamphetamine (PCA) or the serotonin synthesisinhibitor p-chlorophenylalanine (PCPA) leads to an increase inCYP1A activity and a decrease in CYP2C11 and CYP3A activities(Kot and Daniel, 2011). On the other hand, a 3-week tryptophan-freediet enhances the activity of many P450 isoforms (CYP1A, 2A, 2B,2C6, 2D, 3A) and diminishes CYP2C11 activity (Kot et al., 2012).Such changes in cytochrome P450 activity may be due to thereduction in serotonin level in both the brain and periphery and,possibly, due to some additional effects in the liver produced by theapplied substances or the diet. To recognize the effect of the brainserotonergic system alone on the activity of liver cytochrome P450and engagement of central neuroendocrine regulation, all of theaforementioned peripheral influences of neurotoxins or diet have to beexcluded.The aim of the present study was to assess the role of the brain

serotonergic system in the regulation of liver cytochrome P450. Tothis end, we injected the specific serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the DRN and MRN of rat brain,which project to the hypothalamus. Afterward, brain serotonin, serumhormones, and liver cytochrome P450 expression were examined.

Materials and Methods

Animals. Adult male Wistar Han rats (Charles River Laboratories, Sulzfeld,Germany) weighing 280–300 g were kept individually under standardlaboratory conditions (12:12-hour light/dark cycle; temperature of 22 6 2�C;room humidity of 556 5%). The animals had free access to food and tap water,but 18 hours before decapitation, they were deprived of food (both control andneurotoxin-treated animals) because the digestive process might have affectedenzymatic activity. All procedures were carried out in accordance with theNational Institutes of Health Guide for the Care and Use of Laboratory Animals.The protocol was approved by the Bioethical Committee at the Institute ofPharmacology, Polish Academy of Sciences, Kraków.

Drugs and Chemicals. The following compounds were used for the study:noradrenaline, dopamine, serotonin (5-hydroxytryptamine), 5-hydroxyindole-acetic acid (5-HIAA), 5,7-DHT (a creatinine sulfate salt), ascorbic acid,NADPH, NADP, glucose-6-phosphate-dehydrogenase and glucose-6-phosphate,and caffeine and its metabolites (theobromine, paraxanthine, theophylline,and 1,3,7-trimethyluric acid), which were purchased from Sigma-Aldrich(St. Louis, MO). Testosterone and its metabolites were provided bySteraloids (Newport, RI). Warfarin was donated by Merck (Darmstadt,Germany), whereas 7-hydroxywarfarin was synthesized at our institute(Daniel et al., 2006). Bufuralol and 1-hydroxybufuralol were a gift from Dr.Y. Funae of the Osaka University (Osaka, Japan). The polyclonal primary anti-rat CYP1A1 antibody, a secondary antibody (anti-IgG), and rat cDNA-expressed P450s were obtained from Gentest Corp. (Woburn, MA). Thepolyclonal primary anti-rat CYP2C11 antibody was purchased from Abcam(Cambridge, UK); the anti-rat CYP3A1 and CYP3A2 antibodies were obtainedfrom Millipore (Temecula, CA). The chemiluminescence reagent LumiGlo kit

came from KPL (Gaithersburg, MD). Enzyme-linked immunosorbent assay(ELISA) kits for the serum hormones (growth hormone and testosterone) werepurchased from DRG MedTek (Warsaw, Poland), and for corticosterone,triiodothyronine (T3), and thyroxine (T4), from Endocrine Technologies (Newark,CA). ELISA kits for interleukin-2 (IL-2) and IL-6 were obtained from R&DSystems (Minneapolis, MN). All of the organic solvents were of high-performance liquid chromatography (HPLC) purity and were supplied by Merck.Ketamine (ketamine hydrochloride) and Sedazin (xylazine hydrochloride) wereobtained from Biowet (Puławy, Poland).

Surgery and Lesion of Brain Serotonergic System. The rats wereanesthetized with ketamine (65 mg/kg i.p.) and xylazine (10 mg/kg i.p.) andwere placed in a Kopf (Tujunga, CA) stereotaxic apparatus. All solutions werefreshly prepared on the days of experimentation. 5,7-DHT (a toxin specific toserotonergic neurons) was dissolved in a 0.9% NaCl with 0.05% ascorbic acidand injected into the DRN and MRN of the brain at a concentration of 10 mg/ml(1 ml infused at a rate of 1 ml/min) into both raphe nuclei (10 mg per raphenucleus). The following coordinates were used (Paxinos and Watson, 2007):AP (anterior-posterior) –7.9, L (lateral) 0.0 from the bregma, and V (ventral)–7.9 (MRN), –5.9 (DRN) from the surface of the dura (one after the other,respectively). The needle was left in place for 5 minutes after injection before itwas slowly withdrawn. Control rats (sham-operated animals, n = 7) weresubjected to the same procedure as the 5,7-DHT–treated group (n = 7), exceptfor the fact that they received vehicle treatment (a 0.9% NaCl + 0.05% ascorbicacid) instead of 5,7-DHT. The placement of the needle was histologicallyverified in a preliminary experiment with three rats. Moreover, the correctnessof injections was confirmed by measuring serotonin levels in brain structures.An adequate decrease (below 50% of the control level) in the serotoninconcentration of 5,7-DHT–injected animals was observed.

Sample Collection. Ten days after the 5,7-DHT lesion, the rats weredecapitated (10—10:30 a.m.), and their livers and brains were immediatelyremoved. The brains were dissected into the cerebellum, hypothalamus,thalamus, nucleus accumbens, striatum, hippocampus, frontal cortex, cortex,brain stem, and medulla oblongata. The liver and brain tissues were frozen ondry ice and stored at –70�C until they were further analyzed. The blood wascollected into tubes and the serum was separated by centrifugation and stored at–20�C. Liver microsomes were prepared by differential centrifugation in20 mM Tris/KCl buffer (pH 7.4) including washing with 0.15 M KCl, as de-scribed previously (Kot and Daniel, 2011).

Analysis of Serotonin, Its Metabolite 5-HIAA, and CatecholaminergicNeurotransmitters in the Brain. Concentrations of endogenous serotonin andits metabolite 5-HIAA were measured by a high-performance liquidchromatography with electrochemical detection according to the previouslydescribed method (Bromek et al., 2013). Moreover, to check the specificity oflesion of the brain serotonergic system by the neurotoxin 5,7-DHT,concentrations of the catecholaminergic neurotransmitters noradrenaline anddopamine were also simultaneously estimated. In brief, the brain structureswere homogenized in ice-cold 0.1 M HClO4. The obtained homogenates werecentrifuged (15,000� g) and the supernatants were filtered off and injected intothe HPLC system. A GOLD-Hypersil analytical column (3 mm, 100 � 3 mm;Thermo Scientific, Waltham, MA), kept at 30�C, was used. The eluentconsisted of 0.1 M KH2PO4, 0.5 mM Na2EDTA, 80 mg/l sodium1-octanesulfonate, and a 4% methanol, and was adjusted to pH 3.7. The flowrate of the eluent was 0.6 ml/min.

Determination of Cytochrome P450 Isoform Activities in LiverMicrosomes. The activity of rat CYP1A was studied by measuring the rateof caffeine metabolism (C-8-hydroxylation and N-demethylation) at a substrateconcentration of 100 mM, 1 mg of microsomal protein/ml, and incubation timeof 50 minutes. Caffeine and its metabolites were analyzed by HPLC with UVdetection, as described previously (Kot and Daniel, 2008). The activity ofCYP2C6 was studied by measuring the rate of warfarin 7-hydroxylation ata substrate concentration of 60 mM, 1 mg/ml microsomal protein, andincubation time of 15 minutes. Warfarin and its metabolite were analyzed byHPLC with fluorescence detection, as described previously (Daniel et al.,2006). The activities of CYP2A, CYP2B, CYP2C11, and CYP3A wereexamined by measuring the rate of P450 isoform–specific reactions: the 7a-,16b-, 2a- and 16a-, 2b- and 6b-hydroxylation of testosterone, respectively, ata testosterone concentration of 100 mM, 1 mg/ml microsomal protein, andincubation time of 15 minutes. The metabolites formed were analyzed by

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HPLC with UV detection, as described previously (Haduch et al., 2006). Theactivity of CYP2D was studied by measuring the rate of bufuralol19-hydroxylation at a substrate concentration of 10 mM, 0.5 mg/ml microsomalprotein, and incubation time of 10 minutes. Bufuralol and its metabolite wereanalyzed by HPLC with fluorescence detection, as described previously (Hiroiet al., 1998).

Western Blot Analysis and ELISA. The protein levels of cytochrome P450isoforms in the liver microsomes of control and 5,7-DHT–treated rats wereestimated using a Western immunoblot analysis. Microsomal proteins, 10 mg(CYP3A1, CYP3A2, and CYP2C11) or 20 mg (CYP1A), were separated by anSDS polyacrylamide gel electrophoresis, transferred onto nitrocellulosemembranes, and then immunodetected and visualized by chemiluminescence,as previously described (Sadakierska-Chudy et al., 2013). The followingprimary antibodies were used: a polyclonal goat anti-rat antibody, raisedagainst CYP1A1 (Gentest), which also recognized the CYP1A2 isoform;a polyclonal rabbit anti-rat CYP2C11 antibody (Abcam); a polyclonal rabbitanti-rat CYP3A1 and anti-rat CYP3A2 antibodies (Millipore). After incubationwith a primary antibody, the blots were incubated with a secondary antibody,

i.e., a horseradish peroxidase–conjugated anti-IgG (Gentest). The rat cDNA-expressed proteins CYP1A2 (5 mg), CYP2C11 (5 mg), CYP3A1 (1 mg), andCYP3A2 (5 mg) (Supersomes; Gentest) were used as standards. P450 proteinbands were quantified with the Luminescent Image analyzer LAS-1000 usingthe Image Reader LAS-1000 (Fuji Film, Tokyo). Serum hormone and cytokinelevels were measured using ELISA kits: growth hormone and testosterone kits(DRG; MedTek); corticosterone, T3, and T4 ELISA kits (EndocrineTechnologies); and IL-2 and IL-6 ELISA kits (R&D Systems). A SynergyMx Monochromator–based Multi-Mode Microplate Reader (Biotek, Winooski,VT) was used to measure the absorbance.

RNA Isolation and Quantitative Reverse-Transcription PolymeraseChain Reaction Polymerase Chain Reaction. Frozen liver (25 mg) washomogenized using the TissueLyser II (2 � 2 minutes at 30 Hz; QiagenValencia, CA), and the total RNA was extracted using a mirVana isolation kit(Life Technologies, Carlsbad, CA) following the manufacturer’s instructions.RNA was eluted with 50 ml of RNase-free H2O (Sigma-Aldrich). The quantityand the quality of RNA were assessed using a NanoDrop 8000 Spectropho-tometer (Thermo Scientific) and agarose gel electrophoresis. RNA sampleswere stored at 270�C until they were further used. The first-strand cDNAproducts were generated using a Transcriptor High Fidelity cDNA SynthesisKit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’srecommendations. In brief, a reverse transcription was performed using 2 mg ofthe total RNA and oligo(dT) primers at a total volume of 20 ml. cDNAsynthesis was carried out at 55�C for 30 minutes and at 85�C for 5 minutes toinactivate the enzyme. Following the reverse transcription, samples were

Fig. 1. The effect of injection of 5,7-DHT into the median and dorsal raphe nucleiof rat brain on the levels of serotonin (5-HT) (A) and its metabolite 5-HIAA (B) inthe following brain structures: the hypothalamus (Ht), hippocampus (Hp), nucleusaccumbens (Na), striatum (St), thalamus (Th), frontal cortex (Fcx), cortex (Cx),brain stem (Bs), medulla oblongata (Mo), and cerebellum (Cb). The data areexpressed as the mean 6 S.E.M. of the control (n = 7) and the 5,7-DHT (n = 6)group. Statistical significance was assessed by Student’s t test and shown as *P #0.05, **P # 0.01, and ***P # 0.001 compared with the control. The control values(pg/mg of tissue) are as follows: 594.7 6 42.2 (Ht), 267.2 6 68.5 (Hp), 352.3 641.7 (Na), 297.4 6 28.8 (St), 446,4 6 33.3 (Th), 514.5 6 16.5 (Fcx), 305.3 6 22.7(Cx), 472.5 6 47.7 (Bs), 678.5 6 87.7 (Mo), and 44.2 6 3.1 (Cb) (A); 2177.1 6228.57 (Ht), 1014.8 6 45.3 (Hp), 1394.3 6 82.5 (Na), 1772.0 6 80.6 (St), 1875.46 192.3 (Th), 1045.2 6 63.2 (Fcx), 893.7 6 47.6 (Cx), 2309.9 6 226.5 (Bs),1241.3 6 148.9 (Mo), and 426.9 6 11.4 (Cb) (B).

Fig. 2. The effect of injection of 5,7-DHT into median and dorsal raphe nuclei of ratbrain on dopamine (DA) (A) and noradrenaline (NA) (B) levels in the followingbrain structures: hypothalamus (Ht), hippocampus (Hp), nucleus accumbens (Na),striatum (St), thalamus (Th), frontal cortex (Fcx), cortex (Cx), brain stem (BS),medulla oblongata (MO), and cerebellum (Cb). The data are expressed as themean 6 S.E.M. of the control (n = 7) and the 5,7-DHT (n = 6) group. Statisticalsignificance was assessed by Student’s t test and shown as *P # 0.05, **P # 0.01,and ***P # 0.001 compared with the control. The control values (pg/mg of tissue)are as follows: 241.2 6 24.0 (Ht), 14.4 6 4.9 (Hp), 4535.8 6 251.4 (Na), 6553.8 6331.6 (St), 115.96 22.6 (Th), 291.76 55.4 (Fcx), 377.46 43.1 (Cx), 116.16 13.5(Bs), and 36.4 6 4.4 (Mo) (A); 2368.4 6 120.5 (Ht), 286.5 6 17.4 (Hp), 311.2 649.6 (Na), 144.1 6 13.5 (St), 422.7 6 37.6 (Th), 215.4 6 12.8 (Fcx), 169.6 6 14.5(Cx), 501.5 6 26.3 (Bs), 467.3 6 53.3 (Mo), and 139.0 6 9.9 (Cb) (B).

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diluted with 20 ml of RNase-free water and stored at 220�C until subsequenttesting. The expression of genes encoding cytochrome P450 enzymes(CYP2C11, CYP3A1, CYP3A2, CYP1A1, and CYP1A2) and GAPDH andb-actin as reference genes was detected by a real-time polymerase chainreaction (PCR). The reaction mixture, 10 ml, consisted of 4.5 ml of cDNA, 5 mlof TaqMan Gene Expression Master Mix, and 0.5 ml of TaqMan assay (LifeTechnologies, Carlsbad, CA). Negative control samples were processedlikewise, except for the fact that the template was omitted. Real-time PCRruns were performed using the CFX96 PCR system (Bio-Rad, Hercules, CA),and standard thermal cycling conditions were used (50�C for 2 minutes, 95�Cfor 10 minutes, followed by 40 cycles of 95�C for 15 minutes and 60�C for1 minute). The PCR reaction of target and reference genes was run in duplicate.

Real-Time PCR Data Processing. To obtain Ct values (Ct is defined as thecycle number at which the fluorescence crossed the fixed threshold),appropriate thresholds were drawn manually (3000). The mean Ct anda standard deviation were calculated for the replicates of each reaction. Sampleswith replicate standard deviations .0.4 were repeated or excluded from theanalysis. The level of P450 transcripts was normalized to the GAPDH andb-actin expression in each sample, and relative quantification was obtainedusing the comparative delta-delta Ct method (22ΔΔCt). The relative amount oftarget transcript was expressed as a fold change in the expression level relativeto the calibrator (i.e., the average DCt of the control group).

Data Analysis. The obtained values are the mean 6 S.E.M. of five to sevenanimals. Changes in brain neurotransmitter levels, serum hormone, and interleukinconcentrations, as well as liver cytochrome P450 isoform activities, protein, andmRNA levels were statistically assessed using a two-tailed Student’s t test. Theresults were regarded as statistically significant when P , 0.05.

Results

The Effect of Intracerebral Injection of the Neurotoxin 5,7-DHTon the Concentration of Serotonin and Its Metabolite 5-HIAA inthe Brain. Injection of the serotonergic neurotoxin 5,7-DHT into theanterior raphe nuclei DRN and MRN of the brain specifically andsignificantly decreased the level of serotonin and its metabolite 5-HIAAin the brain structures tested (Fig. 1) except for the cerebellum, in which5-HIAA was not changed. The neurotoxin also slightly reduceddopamine levels in the medulla oblongata (Fig. 2A) and noradrenalinelevels in the brain stem (Fig. 2B).The Effect of Intracerebral Injection of the Neurotoxin 5,7-DHT

on P450 Isoform Activities in the Liver. Intracerebral injection of theneurotoxin 5,7-DHT produced statistically significant increases in the

activity of liver CYP2C11 and CYP3A (measured as a rate oftestosterone hydroxylation in position 2a and 16a, or 2b and 6b,respectively); a similar tendency was reported in the case of CYP2B(Fig. 3). The activity of CYP1A increased as the rate of the C-8hydroxylation of caffeine went up (Fig. 4). Such a reaction is specificto rat CYP1A, since at the substrate concentration used (100 mM) thecontribution of CYP1A to this reaction amounts to ca. 73%, whereasthat of CYP3A amounts only to ca. 15% (Kot and Daniel, 2008).Furthermore, CYP1A contributes to 1-N-, 3-N-, and 7-N-demethylation,although to a lower degree, i.e., to ca. 44%, 48%, and 17%, respectively(in the latter case, CYP2C contributes to 65%). The rate of allN-demethylation reactions increased after 5,7-DHT. However, in thecase of 1-N- and 3-N-demethylation, those increases did not reach thelevel of statistical significance (P = 0.11 and P = 0.06, respectively).The activity of CYP2A, CYP2C6, and CYP2D remained practicallyunchanged (Figs. 3 and 4).The Effect of Intracerebral Injection of the Neurotoxin 5,7-DHT

on P450 Isoform Expression in the Liver. The increases in theactivity of CYP1A, CYP2C11, and CYP3A positively correlated withthose in P450 protein levels (Fig. 5). Intracerebral 5,7-DHTsignificantly increased the protein levels of CYP1A, CYP2C11, andCYP3A1 up to 134%, 138%, and 592% of the control, respectively,although not affecting that of CYP3A2, which corresponded to mRNAlevel. The quantitative real-time PCR analyses of P450 genes showeda significant elevation in the mRNA levels of CYP1A1, CYP1A2,CYP2C11, and CYP3A1 (Table 1).The Effect of Intracerebral Injection of the Neurotoxin 5,7-DHT

on the Serum Concentration of Hormones and Cytokines. TheELISA analysis revealed significant increases in the serum concen-tration of the growth hormone, corticosterone, and testosterone, aswell as a decrease in the thyroid hormone T3 level evoked by

Fig. 3. The effect of injection of 5,7-DHT into the median and dorsal raphe nucleiof rat brain on different P450 isoform activites, measured as a rate of specificmetabolic reactions of testosterone in liver microsomes: testosterone 7a- (CYP2A),16b- (CYP2B), 2a- and 16a- (CYP2C11), and 6b- and 2b-hydroxylation (CYP3A).All values are the mean 6 S.E.M. of the control (n = 7) and 5,7-DHT (n = 6) group.Statistical significance was assessed by Student’s t test and indicated as *P # 0.05,**P # 0.01, and ***P # 0.001 compared with the control. The control values(nmol/mg protein/min) are as follows: 152.5 6 9.8, 14.0 6 1.1, 922.4 6 119.7,492.1 6 80.1, 47.6 6 9.3, and 48.5 6 65.2 (testosterone 7a-, 16b-, 2a-, 16a-, 2b-,and 6b-hydroxylation, respectively).

Fig. 4. The effect of injection of 5,7-DHT into the median and dorsal raphe nucleiof rat brain on different P450 isoform activities, measured as a rate of P450 isoform–

catalyzed reactions in liver microsomes: caffeine 1-N-demethylation (CYP1A,CYP2C, and CYP3A), 3-N-demethylation (CYP1A and CYP2C), 7-N-demethylation(CYP1A, CYP2C11, and CYP3A), 8-hydroxylation (CYP1A and CYP3A), warfarin7-hydroxylation (CYP2C6), and bufuralol 19-hydroxylation (CYP2D). All valuesare the mean 6 S.E.M. of the control (n = 7) and the 5,7-DHT group (n = 7).Statistical significance was assessed by Student’s t test and is shown as *P # 0.05,**P # 0.01, and ***P # 0.001 compared with the control. The control values(pmol/mg protein/min) are as follows: 0.8 6 0.09 (caffeine 1-N-demethylation),1.8 6 0.14 (caffeine 3-N-demethylation), 1.9 6 0.19 (caffeine 7-N-demethylation),17.2 6 1.02 (caffeine 8-hydroxylation), 3.6 6 0.38 (warfarin 7-hydroxylation, 7-OH), and1.6 6 0.64 (bufuralol 19-hydroxylation, 19-OH).

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intracerebral injection of the neurotoxin 5,7-DHT. The concentrationsof T4, IL-2, and IL-6 were not changed by 5,7-DHT (Fig. 6).

Discussion

Our earlier studies carried out after intracerebral administration ofcatecholaminergic (dopaminergic and noradrenergic) neurotoxinsshowed that the brain nervous system may influence liver cytochromeP450 expression via central neuroendocrine regulation involving thehypothalamus and pituitary (Wójcikowski et al., 2007, 2008; Bromeket al., 2013; Sadakierska-Chudy et al., 2013; Kot et al., 2015). Ourrecent studies into general damage to the central and peripheralserotonergic (indoleaminergic) systems have shown that serotonin isalso important for the regulation of liver cytochrome P450. Intra-peritoneal administration of the serotonergic neurotoxin PCA or theserotonin synthesis inhibitor PCPA, or a 3-week tryptophan-free dietaffects cytochrome P450 activity in the liver (Kot and Daniel, 2011; Kotet al., 2012).The results presented herein, obtained after intracerebral injection

of the serotonergic neurotoxin 5,7-DHT, indicate that the brainserotonergic system is involved in the regulation of cytochrome P450expression in the liver. Damage to the DRN and MRN of the brain thatproject to the forebrain selectively decreases serotonin levels in allbrain structures studied, including the hypothalamus, followed bychanges in the serum level of hormones regulating (via their receptors)cytochrome P450 expression, which, consequently, leads to anincrease in mRNA and protein levels and the activity of hormone-dependent P450 isoforms in the liver (Fig. 7). This is the first reportshowing that selective damage to the brain serotonergic system affectsliver cytochrome P450.As has been mentioned earlier, after 5,7-DHT lesion of the raphe

nuclei, the concentration of serotonin in the hypothalamus sub-stantially decreases. In turn, this phenomenon produces a rise in theserum levels of the growth hormone and corticosterone, which

positively regulate liver cytochrome P450 expression (Waxman et al.,1995; Waxman and O’Connor, 2006; Dvorak and Pavek, 2010). Onthe other hand, the lesion decreases the concentration of triiodothy-ronine (the most effective thyroid hormone in the regulation of targetgene transcription in the cell nucleus), but does not affect seruminterleukins (IL-2 and IL-6). Both triiodothyronine and the in-vestigated interleukins are known to negatively regulate differentP450 isoforms (Yamazoe et al., 1989; Murayama et al., 1991; Liddleet al., 1998; Zidek et al., 2009). As a consequence, the expression ofCYP1A, CYP2C11, and CYP3A1 (mRNA and protein level), as wellas the enzyme activity (measured as a rate of the metabolism ofcaffeine or testosterone) are augmented. These findings may suggestthat the aforementioned P450 isoforms are induced at the transcriptionlevel.The elevated growth hormone is the chief regulator of the male-

specific gene CYP2C11 and plays an important role in the regulationof CYP3A gene transcription (Waxman et al., 1995; Waxman andO’Connor, 2006). Furthermore, the enhanced concentration oftestosterone may indirectly contribute to the growth hormone–induced induction of these P450 isoforms (Waxman and Holloway,2009). Importantly, the direction of changes in serum GH level andGH-governed CYP2C11 expression (activity, protein, and mRNA) isconsistent.The increased expression of CYP3A1 isoform may also be induced

by the elevated serum corticosterone. CYP3A1 is most sensitive tohormonal regulation, whereas the male-specific CYP3A2 isoform isexpressed at the highest constitutive level among CYP3A subfamilyenzymes (Gibson et al., 2002; Jan et al., 2006). The increasedexpression of CYP1A1/2 isoforms may also be ascribed to theenhancement of serum corticosterone level. CYP1A genes aretranscriptionally governed by an aryl hydrocarbon receptor (AhR),which is positively modulated by the physiologic concentrations ofglucocorticoids in the rat (Monostory et al., 2005). Moreover, theobserved increase in the expression of CYP1A isoforms (and of other

Fig. 5. The effect of injection of 5,7-DHT into the medianand dorsal raphe nuclei of rat brain on the protein levels ofCYP1A1/2, CYP2C11, and CYP3A1/2 isoforms in livermicrosomes. Microsomal protein (20 mg in the case ofCYP1A1/2 or 10 mg in the case of CYP2C11, CYP3A1,and CYP3A2) was subjected to a Western immunoblotanalysis. The rat cDNA-expressed CYP1A2, CYP2C11,CYP3A1, and CYP3A2 proteins (Supersomes) were usedas standards (std). (A) The presented results are typical ofthree (in the case of the control) or four (in the case of5,7-DHT–treated rats) separate animals. (B) The data areexpressed as the mean 6 S.E.M. of control (n = 7) and the5,7-DHT (n = 6) group. Statistical significance wasassessed by Student’s t test and is shown as *P # 0.05,**P # 0.01, and ***P # 0.001 compared with the control.

TABLE 1

The effect of intracerebral injection of 5,7-DHT on the mRNA expression level of P450 genes in rat liver

The results are expressed as a fold change in relation to 2 housekeeping genes: the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and b-actin genecontrols. All values are the mean fold change calculated by the comparative 22ΔΔCt method for control (n = 7) and the 5,7-DHT (n = 6) group.

Gene Name Fold Change Relative to GAPDH Gene Statistical Significance Fold Change Relative to b-Actin Gene Statistical Significance

CYP1A1 3.76 ↑* P , 0.05 5.76 ↑* P , 0.05CYP1A2 1.96 ↑* P , 0.05 1.62 ↑* P , 0.05CYP2C11 1.35 ↑ ns 1.68 ↑* P , 0.05CYP3A1 2.71 ↑* P , 0.05 1.69 ↑ nsCYP3A2 1.03 ns 1.05 ns

ns, not significant.*Statistically significant changes.

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investigated P450s) may also be partly due to the reduced con-centration of thyroid hormone (T3), which negatively regulates theseenzymes (Yamazoe et al., 1989). The signaling pathways of AhR andthe thyroid hormone receptor share several coactivators and tran-scription factors (e.g., retinoid X receptor), a phenomenon that maylead to a transcriptional cross-talk between these pathways andalterations in CYP1A gene expression (Brtko and Dvorak, 2011). Thepresent results confirm our earlier observations that the transcription ofthe CYP1A1 gene in the rat is more responsive to hormonalmodulation than is that of the CYP1A2 gene (Sadakierska-Chudyet al., 2013). Our previous study, carried out after intracerebraladministration of the noradrenergic neurotoxin DSP-4 (N-(2-chlor-oethyl)-N-ethyl-2-bromobenzylamine), showed that the neurotoxinincreased serum corticosterone concentration and decreased thyroid T4

levels, with both effects being followed by enhanced CYP1A1expression (an increase in mRNA level). CYP1A2 expression wasnot significantly changed. We assume that increases in corticosteroneconcentration are mainly involved in the observed stimulation ofCYP1A genes after lesion of the brain monoaminergic systems studied,since corticosterone is known to be an important modulator of thegene expression regulated by AhR (Monostory et al., 2009);moreover, changes observed in this hormone are more pronouncedcompared with those in thyroid hormones.The present results, obtained after intracerebral injection of the

serotonergic neurotoxin 5,7-DHT, showing an increased expression ofCYP1A, CYP2C11, and CYP3A, differ from those reported earlierafter peripheral administration of a serotonergic neurotoxin or aftera tryptophan-free diet. Intraperitoneal administration of the neurotoxinPCA or the serotonin synthesis inhibitor PCPA increased CYP1Aactivity and decreased CYP2C11 and CYP3A activities (Kot andDaniel, 2011), whereas a 3-week tryptophan-free diet (i.e., theserotonin precursor–free diet) enhanced the activity of a number ofP450 isoforms (CYP1A, 2A, 2B, 2C6, 2D, and 3A) and simulta-neously diminished CYP2C11 activity (Kot et al., 2012). However,the aforementioned experimental models involved both the central and

the peripheral serotonergic system. It is noteworthy that 90% ofserotonin is produced in enterochromaffin cells located in the gastricmucosa and taken up by platelets that transport it to an appropriatesite, e.g., the adrenal and thyroid glands or the liver, where it can affectthe proliferative capacity of hepatocytes (Ruddell et al., 2008).Therefore, systemic administration of the aforementioned substances(PCA or PCPA) or a tryptophan-free diet must have also affected theperipheral serotonin pool and its action on the peripheral secretoryglands and the liver. In addition, the applied agents or their reactivemetabolites may act directly on the liver via a nonserotonin mechanism(Kot and Daniel, 2011). On the other hand, the tryptophan-free dietdeprived the organism of the essential amino acid, which might haveaffected the synthesis of enzyme protein (Kot et al., 2012). Since ourpresent results, obtained after damage to the brain serotonergic system,are different from those reported after general serotonin reduction, it issuggested that peripheral serotonin may also contribute to the regulationof cytochrome P450 in the liver by affecting the enzyme in a differentway. It has been found that serotonin receptors are also present in thepituitary (Abrahamson et al., 1987; Dinan, 1996; Balsa et al., 1998;Papageorgiou and Denef, 2007), adrenal gland (Lefebvre et al., 1998),thyroid gland (Csaba and Richter, 1975; Lychkova, 2013), and the liver(Ruddell et al., 2008), where they may affect the release of pituitaryhormones (GH, adrenocorticotropic hormone, TSH), corticosterone, or

Fig. 6. The effect of injection of 5,7-DHT into the median and dorsal raphe nucleiof the brain on hormone and cytokine concentrations in rat blood serum. All valuesare the mean 6 S.E.M. of the control (n = 7) and the 5,7-DHT (n = 6) group.Statistical significance was assessed by Student’s t test and indicated as *P # 0.05,**P # 0.01, and ***P # 0.001 compared with the control. The absolute controlvalues were 2.43 6 1.13 ng/ml, 3.87 6 0.21 ng/ml, 43.48 6 9.85 ng/ml, 72.61 69.63 ng/ml, 1.54 6 0.098 ng/ml, 17.89 6 1.57 pg/ml, and 48.33 6 2.05 pg/ml forthe GH, testosterone (TST), corticosterone (CRT), T4, T3, IL-2, and IL-6,respectively.

Fig. 7. The effect of local 5,7-DHT lesion of the raphe nuclei on serotoninconcentration in the hypothalamus, serum hormone levels, and the expression ofliver cytochrome P450 isoforms (scheme). The dorsal and the median raphe nucleiof the brain send their serotonergic projections to the hypothalamus. The injection ofthe serotonergic neurotoxin 5,7-DHT to the raphe nuclei decreases serotonin level inthe hypothalamus, which affects the hypothalamic endocrine system and leads to anenhanced concentration of the GH and corticosterone and a diminished level ofthyroid hormone (T3) in the blood (serum). This, in turn, stimulates the expression ofcytochrome P450 in the liver (CYP1A/2C11/3A). ACTH, adrenocorticotropichormone; CRH, corticotropin-releasing hormone; GHRH, growth hormone–releasing hormone; SRIH, somatostatin; TRH, thyrotropin-releasing hormone; .The circles (dots) in the raphe nuclei and hypothalamus represent serotoninmolecules.

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thyroid hormones (T3, T4), and thus regulate liver biology,respectively.In conclusion, the results obtained after intracerebral injection of the

serotonergic neurotoxin 5,7-dihydroxytriptamine into the raphe nuclei(DRN and MRN) projecting to the forebrain indicate that the brainserotonergic system contributes to the regulation of liver cytochromeP450 via a neuroendocrine mechanism involving the growth hormone,corticosterone, triiodothyronine, and testosterone. The effect of thebrain serotonergic system on the regulation of liver cytochrome P450expression seems to be different from that observed for braincatecholaminergic systems. Although changes in CYP1A are similar,increases in the expression of CYP2C11 and CYP3A isoforms,produced by a lesion of the brain serotonergic system, are inopposition to decreases in the expression of these P450 isoforms foundafter damage to brain dopaminergic or noradrenergic systems (Wójcikowskiet al., 2007; Sadakierska-Chudy et al., 2013). The observed oppositechanges in enzyme activities are consistent with the different profile ofchanges in blood hormone levels, produced by respective lesions of theinvestigated neurotransmitter systems (Wójcikowski and Daniel, 2008;Sadakierska-Chudy et al., 2013).Further studies are in progress to identify the role of individual

serotonergic nuclei and their projections to specific hypothalamicstructures, as well as different types of serotonin receptors involved inthe central neuroendocrine regulation of cytochrome P450. Clarificationof this neuroendocrine regulatory mechanism of cytochrome P450expression seems important because of its possible practical applicationto pharmacology and pharmacotherapy. The isoforms CYP1A, 2C, and3A, affected by the brain serotonergic system, constitute over 50% of thetotal pool of rat or human cytochrome P450 and are engaged in themetabolism of endogenous steroids and numerous drugs of differentclinical applications. A further explanation of the issue in question shouldhelp to foresee changes in cytochrome P450 activity in the liver, inducedby drugs affecting serotonergic neurotransmission (such as antidepres-sants, neuroleptics, and antianxiety and antiobesity drugs), and tofacilitate drawing conclusions about possible changes in cytochromeP450 activity in pathologic states when the functioning of theserotonergic system is altered. It may also be possible to anticipateinteractions between neuroactive drugs with a serotonin profile andendogenous or exogenous substances (including drugs) at the level of theserotonergic regulation of cytochrome P450.

Authorship ContributionsParticipated in research design: Daniel.Conducted experiments: Rysz, Bromek, Haduch, Sadakierska-Chudy.Performed data analysis: Rysz, Daniel.Wrote or contributed to the writing of the manuscript: Daniel.

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Address correspondence to: Władysława Anna Daniel, Institute of Pharmacol-ogy, Polish Academy of Sciences, Smetna 12, 31-343 Kraków, Poland. E-mail:nfdaniel@cyf-kr.edu.pl

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