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Pharmacological Research 66 (2012) 292– 299

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Pharmacological Research

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imultaneous alterations of brain and plasma serotonin concentrations and liverytochrome P450 in rats fed on a tryptophan-free diet

arta Kota, Andrzej Pilca,b, Władysława A. Daniela,∗

Polish Academy of Sciences, Institute of Pharmacology, Smetna 12, 31-343 Kraków, PolandJagiellonian University, Collegium Medicum, Faculty of Health Sciences, Grzegórzecka 20, 31-531 Kraków, Poland

r t i c l e i n f o

rticle history:eceived 20 April 2012eceived in revised form 18 June 2012ccepted 19 June 2012

eywords:rain and plasma serotoninrain-stemypothalamusat liver cytochrome P450nzyme activity and protein levelryptophan-free diet

a b s t r a c t

Our previous study suggested involvement of the brain serotonergic system in the regulation of livercytochrome P450 (CYP). The aim of the present study was to demonstrate simultaneous responsivenessof liver CYP and the peripheral and brain serotonergic systems to a tryptophan deficient diet during threedays and one or three weeks of ingestion. The concentrations of serotonin, noradrenaline, dopamineand their metabolites were measured in blood plasma, the hypothalamus and brain stem of male rats.The enzyme activity and protein levels in the liver were determined for isoforms CYP1A, CYP2A, CYP2B,CYP2C6, CYP2C11, CYP2D and CYP3A. A three-day tryptophan-free diet increased serotonin content inthe hypothalamus (but not in the brain stem or plasma). After one week, the level of serotonin was notchanged in the brain, but was markedly increased in the plasma. A three week tryptophan restrictionsignificantly reduced the concentration of serotonin in the brain and plasma. Changes in CYP2C6 andCYP2C11 (an increase and a decrease, respectively) were maintained throughout the experiment, while

those found in other CYP isoforms varied, which usually resulted in a gradual increase in the enzymeactivity within three weeks. The observed alterations in liver CYPs suggest involvement of both centraland peripheral serotonin in the regulation of liver CYP expression whose mechanism is discussed. Inconclusion, a deficit in tryptophan in the diet may be responsible for very serious food-cytochromeP450 and food–drug metabolism interactions. Interactions of this type may also refer to drugs acting viaserotonergic system.

. Introduction

Tryptophan is one of the essential amino acids of proteinonstruction and a precursor of serotonin (5-hydroxytryptamine,-HT). Serotonin is synthesized in two enzymatic steps. The firsttep is the hydroxylation of tryptophan by tryptophan hydroxylase,ielding 5-hydroxytryptophan. In the second step, the decar-oxylation of 5-hydroxytryptophan by aromatic l-amino acidecarboxylase, serotonin is produced, which is then inactivated

y monoamine oxidase (MAO-A) and aldehyde dehydrogenase to-hydroxyindole acetic acid (5-HIAA).

Abbreviations: CYP, cytochrome P450; CNS, central nervous system; NA, nora-renaline; DA, dopamine; 5-HT, serotonin; DOPAC, dihydroxyphenylacetic acid;VA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid; HPLC, high per-

ormance liquid chromatography; GHRH, growth-hormone-releasing hormone;H, growth hormone; CRH, corticotropin-releasing hormone; ACTH, adrenocorti-otropic hormone; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulatingormone.∗ Corresponding author. Tel.: +48 12 6623266; fax: +48 12 6374500.

E-mail address: nfdaniel@cyf-kr.edu.pl (W.A. Daniel).

043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.phrs.2012.06.009

© 2012 Elsevier Ltd. All rights reserved.

In the central nervous system (CNS), serotonin is a neurotrans-mitter located in serotonergic neurons. Clusters of serotonergicneurons, also known as cell bodies, are associated with the raphenuclei in the brain stem and they project to numerous parts ofthe brain including the hypothalamus, the latter being the cen-ter of neural regulation of pituitary hormone secretion [1]. On theother hand, blood platelets and, to a considerably lesser extent,enterochromaffin cells of the intestine are a major storage site forserotonin outside the CNS. Recent reports indicate a role of plateletserotonin in liver regeneration [2].

Many drugs used to treat psychiatric or eating disorders affectcytochrome P450 (CYP) activity via their action on the CNS. Ourprevious study showed involvement of psychotropic drugs and theCNS in the regulation of liver cytochrome P450 expression [3–7].Thus, two serotonergic agents (p-chloroamphetamine, a serotoner-gic neurotoxin, or p-chlorophenylalanine, an inhibitor of serotoninsynthesis), injected intraperitoneally to rats, decreased the activityof CYP2C11 and CYP3A, and increased that of CYP1A. At the same

time, the applied neurotoxins had no effect on the activity of CYP2A,CYP2C6 and CYP2D [7]. We propose that the brain serotonergic sys-tem is involved in the physiological neuroendocrine regulation ofliver cytochrome P450 expression. However, some direct effects

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roduced by the reactive metabolites of p-chloroamphetamine or-chlorophenylalanine in the liver cannot be excluded. Therefore it

s advisable to continue investigation into the role of serotonin inhe regulation of cytochrome P450 expression using a more naturalay of reducing the neurotransmitter level in the brain.

A tryptophan-deficient diet offers a specific, physiological,on-pharmacological and non-toxic paradigm for studying the con-equences of reduced serotonin function in animals and humans8–10]. Moreover, this experimental model examines the aspect ofryptophan deficit occurring (among deficits in other nutrients) inndividuals on an incorrect diet, or in patients with eating disorders.

The aim of the present work was to demonstrate simultaneousesponsiveness of liver cytochrome P450, as well as blood plasmand brain serotonin to dietary tryptophan manipulations. To thisnd, the rats were fed on a tryptophan-free diet for 3, 7 or 21 days,hich allowed us to observe the development of changes in plasma

nd brain serotonin versus liver cytochrome P450. Compared tour previous study, the present research deals more extensivelyith the regulation of liver cytochrome P450 expression by the ner-

ous system, and gives further support to the hypothesis about thehysiological regulation of cytochrome P450 by the serotonergicystem.

. Materials and methods

.1. Animals

All the experiments with animals were performed in accor-ance with the Polish state regulations (The Animal Protectionct, DZ.U. 97.111.724, 1997). Male Wistar rats (Charles River,anover, Germany) were used; throughout the experiment, theyere kept under standard laboratory conditions (room tempera-

ure of 22 ± 2 ◦C; room humidity of 55 ± 5%; a 12-h light/dark cycle,he light on from 6:00 AM to 6:00 PM). The animals had free accesso water and food.

.2. Animal procedures

The rats were fed on a tryptophan-free diet for 3, 7 or1 days (a tryptophan-free synthetic amino acid diet; MP Biomed-

cals, France). Control animals were on a standard diet (providedy the same company) for 3, 7 or 21 days, respectively, i.e.ach experimental time period had its own control. When thebove-mentioned treatment periods were over, the animals wereecapitated and their brain structures and livers were quicklyemoved and frozen. The brain structures and livers were storedt −80 ◦C. Additionally, the blood samples collected during decap-tation were centrifuged at 2000 × g. The obtained plasma samples

ere stored at −20 ◦C.

.3. Drugs and chemicals

Caffeine and its metabolite 1,3,7-trimethyluric acid, asell as NADPH, noradrenaline (NA), dopamine (DA), 3,4-ihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA),erotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindoleaceticcid (5-HIAA) were purchased from Sigma (St. Louis, USA).arfarin was donated by Merck (Darmstadt, Germany), while 7-

ydroxywarfarin was synthesized in our institute [11]. Bufuralol

nd 1′-hydroxybufuralol were a gift from Dr. Y. Funae of the Osakaniversity, Japan. Testosterone and its metabolites came from Ster-loids (Newport, KY, USA). All the organic solvents were of HPLCurity and were supplied by Merck (Darmstadt, Germany).

search 66 (2012) 292– 299 293

2.4. Preparation of rat liver microsomes

Microsomes were prepared from individual rat livers by differ-ential centrifugation in a 20 mM Tris/KCl buffer (pH = 7.4), includingwashing with 0.15 M KCl according to the method of Legrum et al.[12], a modification of Netter’s procedure [13].

2.5. Determination of monoaminergic neurotransmitters andtheir metabolites

2.5.1. Determination of NA, DA, 5-HT and their metabolites in thebrain

Selected structures contained the initial part (the brain stem)and the endings (the hypothalamus) of serotonergic projections.Those brain structures were homogenized in an ice-cold 0.1 MHClO4. The homogenates were centrifuged at 10,000 × g, and thesupernatants were filtered through a 0.2 �m membrane (Alltech).Immediately after filtration, the samples were analyzed for the con-tent of serotonin and its metabolite 5-HIAA, as well as for the otheraccompanying monoaminergic neurotransmitters NA, DA and theirmetabolites (DOPAC, HVA) using a high performance liquid chro-matography (HPLC) with electrochemical detection [14].

2.5.2. Determination of the plasma levels of serotonin and 5-HIAAThe plasma levels of 5-HT and 5-HIAA were estimated using

HPLC with electrochemical detection as described above.

2.6. Cytochrome P450 activity assay

Studies into caffeine, warfarin and testosterone metabolismwere carried out at the linear dependence of product formationon time, and on the concentration of protein and a substrate.

The activity of CYP1A2 was studied by measuring the rateof caffeine 8-hydroxylation (catalyzed by CYP1A2) at a substrateconcentration of 100 �M as described previously with a minormodification (1.5 mM NADPH replaced with the NADPH generat-ing system); caffeine and its metabolite were analyzed by HPLCwith UV detection [15,16].

The activity of CYP2C6 was studied by measuring the rate ofwarfarin 7-hydroxylation at a substrate concentration of 60 �M asdescribed previously; warfarin and its metabolite were analyzedby HPLC with fluorescence detection [11].

The activities of CYP2A, CYP2B, CYP2C11 and CYP3A were stud-ied by measuring the rate of P450-specific reactions: the 7�-,16�-, 2�- and 16�-, 2�- and 6�-hydroxylation of testosterone,respectively, at a substrate concentration of 60 �M, as describedpreviously; testosterone and its metabolites were analyzed by HPLCwith UV detection [17–19].

The activity of CYP2D was studied by measuring the rate of bufu-ralol 1′-hydroxylation at a substrate concentration of 10 �M usingHPLC with a fluorescence detector, as described previously [20].

2.7. Western blot analysis

The protein levels of CYP1A, CYP2B, CYP2C6, CYP2C11 andCYP3A isoforms in the liver microsomes of control rats and animalsfollowing a tryptophan-free diet were estimated using Westernimmunoblot analyses as described previously [11,19]. Microso-mal proteins, 10 �g, were separated by an SDS polyacrylamidegel electrophoresis on a 12% separating gel according to Laemmli[21], and employing a MINIPROTEAN II electrophoresis system(Bio-Rad, Hemmel Hempstead, UK; 130 V, 65 min). The following

primary antibodies for liver microsomal CYPs were used: a poly-clonal goat anti-rat antibody raised against CYP1A1, which alsorecognized the CYP1A2 isoform; a polyclonal goat anti-rat anti-body raised against CYP2B1, which also recognized the CYP2B2

294 M. Kot et al. / Pharmacological Research 66 (2012) 292– 299

Fig. 1. The levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus during ingestion of a tryptophan-free diet. All the values are the mean ± SEM (n = 6). Statistical significance wasa paredH eek try

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ssessed by Student’s t-test and indicated by ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 comVA and 5-HIAA (pg/mg of tissue), referring to a three-day, one-week and three-w

soform (Gentest); a monoclonal mouse anti-rat antibody raisedgainst CYP2C6 (Abcam); a polyclonal rabbit anti-rat antibodyaised against CYP2C11, which recognized to a lesser extent theYP2C6 and CYP2C13 isoforms, and a polyclonal rabbit anti-ratntibody raised against CYP3A2, which also recognized the CYP3A1soform (Gentest). After incubation with a primary antibody, blots

ere incubated with a secondary antibody, e.g. an appropriatepecies-specific horseradish peroxidase-conjugated anti-IgG. RatDNA-expressed CYP1A1/2, CYP2B1, CYP2C6, CYP2C11 and CYP3A2soforms (Supersomes) were used as standards. Bands on the nitro-ellulose membrane were quantified with the Luminescent Imagenalyzer LAS-1000 using the Image Reader LAS-1000 and Imageauge 3.11 programs (Fuji Film, Japan).

.8. Statistical analysis

Statistical significance was assessed using Student’s t-test. Allhe obtained values are the mean ± SEM from 6 animals per group.

. Results

.1. The effect of a tryptophan-deficient diet on the content of NA,A, 5-HT and their metabolites in brain structures

A three-day tryptophan-restricted diet significantly increasedhe content of serotonin and its metabolite 5-HIAA in the hypotha-amus (Fig. 1), but not in the brain stem (Fig. 2). After one-weekeeding on a tryptophan-free diet, the levels of monoaminergic neu-otransmitters did not change in the hypothalamus or the braintem (Figs. 1 and 2). A three-week tryptophan-restricted diet sig-

ificantly reduced the concentration of serotonin and its metabolite-HIAA and DA in the hypothalamus (Fig. 1). At the same time, theoncentrations of serotonin and 5-HIAA, as well as of the dopamineetabolites DOPAC and HVA decreased in the brain stem (Fig. 2).

to the appropriate control. The individual control values of NA, DA, 5-HT, DOPAC,ptophan-deficient diet, are presented in supplementary Table.

3.2. The effect of a tryptophan-deficient diet on the content of5-HT and its metabolite 5-HIAA in the plasma

A three-day tryptophan deprivation in the diet did not affectthe levels of serotonin and its metabolite 5-HIAA (Fig. 3) in theplasma. One-week feeding on a tryptophan-free diet led to an enor-mous increase in the concentration of serotonin in the plasma(Fig. 3). Three weeks of tryptophan restriction produced a profounddecrease in the content of serotonin and its metabolite 5-HIAA inthe plasma (Fig. 3).

3.3. The effect of a tryptophan-deficient diet on CYP activity inthe liver

The rats fed on a tryptophan-deficient diet did not put onweight as did control animals, and after three weeks their livermass fell to 62% of the control value. A three-day tryptophan-free diet decreased the activity of CYP2C11 (testosterone 2�-and 16�-hydroxylation), but increased that of CYP2B (testos-terone 16�-hydroxylation), CYP2C6 (warfarin 7-hydroxylation)and CYP3A (testosterone 2�- and 6�-hydroxylation) (Fig. 4A). Aone-week tryptophan-free diet significantly diminished the activ-ity of CYP2B and CYP2C11. At the same time, the activity of CYP2A(testosterone 7�-hydroxylation), CYP2C6 and CYP3A was enhanced(Fig. 4A). Three-week feeding on a tryptophan-restricted diet sig-nificantly reduced the activity of CYP2C11 only. In contrast, theactivity of the other CYP isoforms tested, i.e. CYP1A2 (caffeine8-hydroxylation), CYP2A, CYP2B, CYP2C6, CYP3A and CYP2D (bufu-ralol 1′-hydroxylation) was enhanced (Fig. 4A).

3.4. The effect of a tryptophan-deficient diet on CYP protein levelin the liver

Three-day feeding on a tryptophan-free diet significantlyreduced the level of CYP2C11 protein only, while the amount ofCYP1A and CYP2C6 increased (Fig. 4A and B). The protein level of

M. Kot et al. / Pharmacological Research 66 (2012) 292– 299 295

Fig. 2. The levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-h n-freeb the a5 phan

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ydroxyindoleacetic acid (5-HIAA) in the brain stem during ingestion of a tryptophay Student’s t-test and is indicated by ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 compared to-HIAA (pg/mg of tissue), referring to a three-day, one-week and three-week trypto

YP2B and CYP3A was similar to that of the control. After one-eek feeding on a tryptophan-free diet, the level of CYP2C11 fell,hereas that of CYP1A, CYP2C6 and CYP3A rose. The protein level of

YP2B showed a decreasing tendency. Changes in CYP1A, CYP2C11nd CYP3A were more pronounced after a three-week tryptophan-ree diet. A significant decrease in CYP2C11 protein level and a

ig. 3. The plasma levels of serotonin (5-HT) and its metabolite 5-ydroxyindoleacetic acid (5-HIAA) during ingestion of the tryptophan-freeiet. All the values are the mean ± SEM (n = 6). Statistical significance was assessedy Student’s t-test and is indicated by ***p ≤ 0.001 compared to the appropriateontrol. The individual control values of 5-HT and 5-HIAA (pg/ml), referring to ahree-day, one-week and three-week tryptophan-deficient diet, are presented inupplementary Table.

diet. All the values are the mean ± SEM (n = 6). Statistical significance was assessedppropriate control. The individual control values of NA, DA, 5-HT, DOPAC, HVA and-deficient diet, are presented in supplementary Table.

spectacular rise in CYP1A and CYP3A protein levels were observed.The level of CYP2C6 protein rose, while that of CYP2B showed anincreasing tendency only.

4. Discussion

Our study describes the relationship between serotonin con-centration in the brain and in plasma on the one hand, and livercytochrome P450 activity on the other at three time-points of thetryptophan-deficient diet. Among the investigated rat CYP isoformsthere were counterparts of the main human drug-metabolizingenzymes in the liver (e.g. rat CYP1A2 and human CYP1A2, ratCYP2C6/11 and human CYP2C9, rat CYP2D1/2 and human CYP2D6,or rat CYP3A1/2 and human CYP3A4) [16,22–25].

When a tryptophan-free diet was followed for three days, avisible increase in the concentration of serotonin and its metabo-lite 5-HIAA could be seen in the hypothalamus (but not in thebrain stem), being a possible natural response to the decreas-ing amount of the substrate (tryptophan) in the blood. By actingon its receptors in the hypothalamus, serotonin can regulate thesynthesis and secretion of some releasing hormones such as, e.g.GHRH [26–28], CRH [29,30] or TRH [31], via 5-HT2A-, 5-HT2C-or 5-HT1A receptors. The main 5-HT2A- and 5-HT2C receptor-G protein-coupled intracellular pathways activate phospholipase,followed by a breakdown in phosphatidylinositol and generation ofsecondary messengers, which leads to activation of protein kinasesand, in consequence, stimulation of GHRH- or CRH-gene expres-sion in the hypothalamus. GHRH and CRH stimulate the secretionof anterior pituitary hormones (GH, ACTH); in this way they posi-tively regulate cytochrome P450 expression: directly – via GH, orindirectly – via corticosterone, respectively. The serotonin regu-lation of thyroid hormones (at the TRH or TSH level) has not yetbeen fully explained [31,32]. By acting on their receptors (GHR, GR,

TR), respective hormones (GH, cortisterone or thyroid hormones,respectively) activate numerous signaling pathways, thus inter-acting with different transcription factors and nuclear receptorsinvolved in the regulation of liver CYP gene expression [33–37].

296 M. Kot et al. / Pharmacological Research 66 (2012) 292– 299

Fig. 4. (A) The influence of the tryptophan-deficient diet on different CYP isoform activities and protein levels. The activities were measured as rates of the CYP isoform-specific reaction in rat liver microsomes: caffeine 8-hydroxylation (CYP1A2), testosterone 7�-hydroxylation (CYP2A), testosterone 16�-hydroxylation (CYP2B), warfarin7-hydroxylation (CYP2C6), bufuralol 1′-hydroxylation (CYP2D), testosterone 2�- and 16�-hydroxylation (CYP2C11), testosterone 2�- and 6�-hydroxylation (CYP3A). Theindividual control values (pmol/mg of protein/min) are as follows: a three-day tryptophan-deficient diet: 14.5 ± 2.2 (caffeine 8-hydroxylation), 6.4 ± 0.5 (warfarin 7-hydroxylation), 14.6± 2.8 (bufuralol 1′-hydroxylation), 167.3 ± 9.9, 28.9 ± 2.4, 1882.9 ± 186.3, 2397.8 ± 241.5, 93.7 ± 5.6, 666.1 ± 30.8 (testosterone 7�-, 16�-, 2�-, 16�-,2�-, 6�-hydroxylation, respectively); a one-week tryptophan-deficient diet: 9.0 ± 0.6 (caffeine 8-hydroxylation), 7.1 ± 0.5 (warfarin 7-hydroxylation), 18.4 ± 2.1 (bufuralol1′-hydroxylation), 153.8 ± 9.9, 105.6 ± 17.0, 1805.9 ± 134.9, 2065.8 ± 253.6, 73.1 ± 1.7, 554.7 ± 71.5 (testosterone 7�-, 16�-, 2�-, 16�-, 2�-, 6�-hydroxylation, respectively); athree-week tryptophan-deficient diet: 5.4 ± 1.9 (caffeine 8-hydroxylation), 7.2 ± 0.5 (warfarin 7-hydroxylation), 29.2 ± 1.4 (bufuralol 1′-hydroxylation), 109.1 ± 7.3, 20.3 ± 2.3,1097.4 ± 114.5, 1393.9 ± 150.6, 60.7 ± 6.6, 387.5 ± 57.0 (testosterone 7�-, 16�-, 2�-, 16�-, 2�-, 6�-hydroxylation, respectively). All the values are the mean ± SEM (n = 6).Statistical significance was assessed by Student’s t-test and is indicated by ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 compared to the appropriate control. (B) The influence of thetryptophan-deficient diet on the protein levels of CYP1A, CYP2B, CYP2C6, CYP2C11 and CYP3A isoforms in rat liver microsomes. Microsomal proteins, 10 �g, were subjectedto a Western immunoblot analysis. Rat cDNA-expressed CYP1A1/2, CYP2B1, CYP2C11 and CYP3A2 isoforms (Supersomes) were used as standards. The presented results aretypical of three (for the control) and four (for samples collected after the diet) animals. The data expressed as the mean ± SEM (n = 6) are shown in Fig. 4A.

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At the same time, the activity of liver isoforms CYP2B, CYP2C6nd CYP3A is enhanced, while that of CYP2C11 is reduced (thectivity of CYP2C6 and 2C11 correlates positively with the enzymerotein level). On the other hand, the activity of CYP1A2, CYP2And CYP2D isoforms (less susceptible to direct hormonal regula-ion) is not affected [38]. Since the level of serotonin is not changedn the plasma, it is postulated that modifications of CYP activ-ty in the liver, observed after a three-day tryptophan-deficientiet, stem from the central neuroendocrine regulation controlledy hypothalamic serotonin [6,7]. However, if this is the case,YP2C11 (a predominant male isoform), which is positively reg-lated by the growth hormone [35,39,40], should undergo up-,ut not down-regulation (as observed), since central serotonin isnown to stimulate growth hormone secretion [41–43]. Thereforeome role of hypothalamic serotonin in central autonomic regula-ion, or that of tryptophan in the immunoregulation of liver CYPannot be excluded [6,7]. The expression of several CYP isoforms isuppressed by such cytokines as interleukin-6 (IL-6), interleukin-� (IL-1�) and interleukin-2 IL-2 [44], while tryptophan has beenound to reduce the plasma levels of proinflammatory cytokines45]. On the other hand, the decreased plasma tryptophan level maydditionally affect cytochrome P450 expression in a more directay (not only via the nervous system). It has been shown that tryp-

ophan or its indole derivatives increase hepatic protein synthesis,ytochrome P450 content, and the enzyme activity of rats and otherpecies in vitro and in vivo [46–51]. Moreover, tryptophan is anmino acid which is essential for the synthesis of cytochrome P450.ertain tryptophan residues (a few of which are preserved in mam-alian cytochrome P450) have been found to be of both structural

nd functional importance to the enzyme [52,53].After a one-week tryptophan-deficient diet, a transition from

he regulation by the CNS to the peripheral regulation of theytochrome took place. The level of serotonin apparently stabi-ized in the brain, while a spectacular increase in its concentration

as observed in the plasma. The above-described effect may beue to a massive release of serotonin from blood platelets. It isell known that serotonin is abundantly accumulated and stored

n blood platelets, hence even subtle enhancement of its releaserom this pool may lead to a dramatic increase in its concentra-ion in the plasma [54]. The observed elevation of plasma serotoninndicates the onset of peripheral regulation of liver CYP activity byerotonin. Thus an increase in the activity of CYP2A and a decreasen that of CYP2B (but not a fall in protein level) were observedompared to a three-day specific diet. The lack of changes in theevel of CYP2B protein suggests posttranslational regulation of thissoform by the circulating serotonin. It seems that the extremelyigh serotonin level in the plasma, observed after a one-weekryptophan-deficient diet, stimulates the 5-HT receptors presentn the liver [55], which – in turn – leads to an increased phos-horylation of CYP2B and its inactivation. The above assumption

s in line with the fast regulation of CYP2B via phosphorylation,s observed by Oesch-Bartlomowicz and Oesch [56]. On the otherand, the decreased activity of CYP2C11 and the increased activityf CYP2C6 and CYP3A, observed after a three-day tryptophan-ree diet, could still be seen after one week (they were moreronounced at that time), and were positively correlated withhanges in enzyme protein levels. Although the total concentra-ion of serotonin in the hypothalamus and brain stem was nothanged at that time point, this finding does not exclude anydditional brain-derived effects, since serotonin (elevated in thelasma) can penetrate the eminentia mediana of the brain (a struc-ure devoid of the blood–brain-barrier) and be then transported

y hypophyseal portal blood to the anterior pituitary lobe where

t can modulate the secretion of its hormones via serotonergiceceptors [57–60]. The serotonin receptors present in the anterioritutitary and adrenal cortex can directly stimulate the release of

search 66 (2012) 292– 299 297

ACTH and corticosterone, respectively (via 5-HT1A and 5-HT2A, and5-HT2C receptors, respectively), irrespective of CRH [61]. Pituitaryserotonin receptors also stimulate GH release (mainly via 5-HT2Breceptors) [62,63] and, probably, TSH release (via 5-HT2 receptors)[31].

A three-week tryptophan-deficient diet, which simulated theonset of a chronic condition, developed a wide spectrum of conse-quences (presumably including adaptive changes), visible in thebrain, plasma and liver. The concentrations of serotonin and itsmetabolite 5-HIAA fell in both the brain structures tested and theplasma. Furthermore, a significant loss of dopamine in the hypotha-lamus and of its metabolites DOPAC and HVA in the brain stemwas observed. Since aldehyde dehydrogenase takes part in themetabolism of both serotonin (to 5-HIAA) and dopamine (to DOPACand HVA), the adaptive change in the activity of this enzyme, pro-duced by a decrease in serotonin concentration, may affect bothindole and catechol metabolites [64,65]. Moreover, anatomical con-nections and functional interactions between the serotonin anddopamine systems may also cause changes in the level of dopamineand its metabolites in the brain under conditions of serotonin deficit[66–69].

Parallel to the reduced serotonin levels in the brain and plasma,a prolonged absence of tryptophan in the diet produced hugeincreases in the activity of the liver CYP isoforms studied (inparticular CYP1A, CYP2A, CYP2B, CYP2C6 and CYP3A), except forCYP2C11 whose activity remained decreased. The observed effecton the enzyme activity, produced by a three-week tryptophan-freediet, corresponded well with the accompanying alterations in therespective enzyme protein levels, except for the concentration ofCYP2B. As discussed elsewhere, the diminished stimulation of 5-HT receptors in the liver may prevent CYP2B phosphorylation andinactivation [56]. The changes in liver CYP isoforms, observed aftera three-week tryptophan-free diet, seem to result from simulta-neous alterations in the functioning of the central neuroendocrinesystem (produced by a decrease in serotonin and dopamine levelsin the hypothalamus) and the peripheral nervous system (affectingliver serotonin receptors). Both those systems became deficient inserotonin which, in turn, may have led to adaptive changes in theirfunctioning [70,71]. Moreover, both brain and peripheral serotonincoordinate food intake [72], the latter being known to modulatecytochrome P450 expression [73–75]. The hypofunction of sero-tonin results in an increased appetite and body weight, as observedin patients suffering from bulimia nervosa (opposite to anorexianervosa) [76,77]. However, rats fed on a tryptofan-free diet did notput on weight as did control animals and their livers diminished,which makes the estimation of a possible food effect on the enzymeactivity difficult in our experiment.

The results of the present study concerning the three-weektryptophan-free diet differ somewhat from our previous find-ings, obtained one week after intraperitoneal administrationof the serotonin-depleting agent p-chloroamphetamine or p-chlorophenylalanine [7], although in both those experimentalprocedures the concentration of serotonin was diminished. In ourprevious experimental model we observed mainly central effects ofthe decreased serotonin level, which resulted in an enhanced activ-ity of CYP1A2 and a diminished activity of CYP2C11 and CYP3A,but no effect on other CYP isoforms [7]. Thus the observed dif-ferences in CYP activity between the two models used may arisefrom the diverse contribution of the peripheral nervous system andthe deficit of tryptophan in the organism, as well as from the pos-sible development of adaptive changes in the functioning of theserotonergic system after a three-week tryptophan-free diet.

In summary, our results show changes in brain and plasmaserotonin levels at three time points of the tryptophan-deficientdiet and some accompanying modifications in liver CYP activ-ity. Interestingly, changes in the isoforms CYP2C6 and CYP2C11

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an increase and a decrease, respectively) were maintainedhroughout the experiment, while those pertaining to other CYPsoforms varied during that period, usually leading to a gradualncrease in the enzyme activity. Since serotonin synthesis, stor-ge/release and metabolism depend on the duration of a diet andre different inside and outside the CNS, diverse mechanisms takeart in the physiological regulation of liver CYP expression dur-

ng ingestion of a tryptophan-free diet. Hence the results of theresent study pose a challenge to future biochemical/molecular

nvestigations aimed at explaining the mechanism responsible forhe observed multiple changes in the response of cytochrome P450o the tryptophan-free diet. However, it should be noted that thepplied diet has certain limitations compared to clinical conditions,.e. it represents a complete lack of one nutrient only. All the same,hanks to it, the experimental model used allows us to contemplatehe consequences of an exclusive deficiency of tryptophan (or itsole) in the diet.

In conclusion, tryptophan deficit in the diet, observed in individ-als fed on an ill-balanced diet or in patients with eating disorderse.g. anorexia nervosa), may be responsible for the very serious

etabolic food-cytochrome P450 and food–drug metabolism inter-ctions. Interactions of this type may also be characteristic of drugscting via the serotonergic system, and may be clinically importantn the case of drugs with a narrow therapeutic concentration range.

onflict of interest

The authors declare that they are not engaged in any conflict ofnterest.

cknowledgements

The study was supported by statutory funds from the Institute ofharmacology, Polish Academy of Sciences (Kraków, Poland), andy grant no. N N405 055737 from the Ministry of Science and Higherducation (Warszawa, Poland). Additionally, thanks are due to Ms.ustyna Starynska for her kind technical assistance.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.phrs.2012.06.009.

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