CYP2D6 increases toxicity of the designer drug 4-methylthioamphetamine (4-MTA).

Abstract

4-Methylthioamphetamine (4-MTA) belongs to a group of new amphetamine derivatives that is usually sold as "ecstasy" or "flatliners" on the illicit drug market. Large interindividual differences in 4-MTA mediated toxicity have been reported in humans. Therefore, we tested whether CYP2D6 or its variant alleles as well as CYP3A4 influence the susceptibility to 4-MTA. For this purpose, we used the colony formation assay with Chinese hamster lung fibroblast V79 cells expressing human wild-type CYP2D6 (CYP2D6*1), the low activity alleles CYP2D6*2, CYP2D6*9, as well as human CYP3A4. The obtained results showed that the expression of wild type CYP2D6*1 clearly enhanced the susceptibility to the cytotoxic effects of 4-MTA compared with the parental cells devoid of CYP-dependent enzymatic activity. Toxicity in V79 CYP2D6*1 was also higher compared to the V79 cell lines expressing the low activity alleles CYP2D6*2 and CYP2D6*9. In contrast to CYP2D6, the CYP3A4 isoenzyme did not enhance 4-MTA toxicity. In conclusion, our results suggest that CYP2D6 rapid metabolizers may be more susceptible to 4-MTA toxicity than CYP2D6 poor metabolizers.

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Toxicology 229 (2007) 236–244
CYP2D6 increases toxicity of the designer drug
4-methylthioamphetamine (4-MTA)
Helena Carmoa,∗, Marc Brulportb, Matthias Hermesb, Franz Oeschc,
Douwe de Boerd, Fernando Remi˜ aoa, F´ elix Carvalhoa,
Michael R. Sch¨ one, Niels Krebsfaengerf, Johannes Doehmerg,
Maria de Lourdes Bastosa, Jan G. Hengstlerb
aREQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Rua An´ ıbal Cunha 164,
4099-030 Porto, Portugal
bCentre for Toxicology, University of Leipzig, Hartelstrasse, 16-18, 04107 Leipzig, Germany
cInstitute of Toxicology, University of Mainz, Germany
dDepartment of Clinical Chemistry, University Hospital Maastricht, The Netherlands
eDepartment of Surgery, University of Leipzig, Germany
fSchwarz Biosciences GmbH, Department of Pharmacology and Toxicology, Monheim, Germany
gGenPharmTox BioTech AG, D-Planegg/Martinsried, Germany
Received 1 September 2006; received in revised form 25 October 2006; accepted 27 October 2006
Available online 5 December 2006
Abstract
4-Methylthioamphetamine(4-MTA)belongstoagroupofnewamphetaminederivativesthatisusuallysoldas“ecstasy”or“flatlin-
ers”ontheillicitdrugmarket.Largeinterindividualdifferencesin4-MTAmediatedtoxicityhavebeenreportedinhumans.Therefore,
wetestedwhetherCYP2D6oritsvariantallelesaswellasCYP3A4influencethesusceptibilityto4-MTA.Forthispurpose,weused
the colony formation assay with Chinese hamster lung fibroblast V79 cells expressing human wild-type CYP2D6 (CYP2D6*1), the
lowactivityallelesCYP2D6*2,CYP2D6*9,aswellashumanCYP3A4.Theobtainedresultsshowedthattheexpressionofwildtype
CYP2D6*1 clearly enhanced the susceptibility to the cytotoxic effects of 4-MTA compared with the parental cells devoid of CYP-
dependentenzymaticactivity.ToxicityinV79CYP2D6*1wasalsohighercomparedtotheV79celllinesexpressingthelowactivity
allelesCYP2D6*2andCYP2D6*9.IncontrasttoCYP2D6,theCYP3A4isoenzymedidnotenhance4-MTAtoxicity.Inconclusion,
our results suggest that CYP2D6 rapid metabolizers may be more susceptible to 4-MTA toxicity than CYP2D6 poor metabolizers.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: 4-Methylthioamphetamine; Designer drugs; Metabolism; Hepatotoxicity; CYP2D6; Polymorphism
1. Introduction
4-Methylthioamphetamine
a group of new amphetamine derivatives that have
(4-MTA)belongsto
∗Corresponding author. Tel.: +351 222078979;
fax: +351 222003977.
E-mail address: helenacarmo@ff.up.pt (H. Carmo).
emerged on the illicit drug market for recreational drugs
where it is usually sold as tablets marketed as “ecstasy”
or “flatliners”. Since its first identification in 1997 there
have been at least six cases of death associated with
4-MTA in the UK and in the Netherlands (EMCDDA,
1999). In addition, one fatal and seven nonfatal intox-
ications in Belgium have been reported (de Letter et
al., 2001). Therefore, 4-MTA was placed in Schedule I
0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2006.10.024

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H. Carmo et al. / Toxicology 229 (2007) 236–244
237
of the 1971 Convention (WHO, 2001) and is submitted
to control measures and criminal penalties within
the member States of the European Union (European
Communities, 1999; EMCDDA, 1999).
4-MTA acts in a similar way to other amphetamine
derivatives, such as p-methoxyamphetamine and 3,4-
methylenedioxymethamphetamine (MDMA, “ecstasy”)
(Dukat et al., 2002; Khorana et al., 2004). It is a
very selective serotonergic agent, increasing the release
of serotonin, inhibiting serotonin uptake from nerve
endings (Huang et al., 1992), with low affinity for nora-
drenaline and dopamine uptake sites and monoamine
receptors (Huang et al., 1992; Li et al., 1996), and also
inhibitingmonoamineoxidase-Aactivity(Lietal.,1996;
Scorza et al., 1997).
Particular concern regarding the toxicity of 4-MTA
has been raised since its slow onset of action has
encouraged abusers to administer further doses assum-
ing that the first dose was inadequate (EMCDDA,
1999; Kavanagh et al., 1999). In the reported 4-MTA-
related intoxications in humans, symptoms typical for
the sympathomimetic effects of amphetamine-like com-
pounds were observed, such as tachycardia, tremors,
stomach cramps, headache, and sweating (de Boer et
al., 1999; de Letter et al., 2001; Elliott, 2000). Infor-
mation on the toxicity of 4-MTA is still scarce. The
ability of 4-MTA to induce a hyperserotonergic state
known as the serotonin syndrome (which is character-
ized by confusion, fever, shivering, diaphoresis, ataxia,
hyperreflexia, myoclonus, or diarrhea) (Sporer, 1995)
was observed in neurotoxicity studies performed with
rats (Huang et al., 1992). Although initially consid-
ered as a nonneurotoxic drug, in vitro studies showed
that 4-MTA induced neurotoxicity in rat hypothalamic
cultured cells (Hurtado-Guzman et al., 2002). Addition-
ally, in vivo studies in mice have shown that 4-MTA
induces hyperthermia, another potentially lethal con-
sequence of amphetamine intoxications (Carmo et al.,
2003).
The interindividual variation in the propensity to the
adverse effects of amphetamine designer drugs has been
increasingly noticed (de Letter et al., 2004; Henry et al.,
1992; O’Donohoe et al., 1998). Several studies suggest
that interindividual differences in metabolism may be
responsible for these differences (Capela et al., 2006;
Carvalho et al., 2002; Carvalho et al., 2004a,b,c; Easton
et al., 2003; Escobedo et al., 2005; Forsling et al., 2002;
Gollamudi et al., 1989; Hartung et al., 2002; Jones et al.,
2005).
The metabolic pathways proposed for 4-MTA in
humans are represented in Fig. 1 and include: (i) oxida-
tive deamination into a ketone metabolite (deamino-oxo
4-MTA; 1-[4-(methylthio)-phenyl]propan-2-one) that
can be further reduced to the corresponding alcohol
(deamino-hydroxy 4-MTA; 1-[4-(methylthio)-phenyl]
propan-2-ol) or suffer degradation of the side chain
into the 4-methylthiobenzoic acid metabolite; (ii) ring
hydroxylation to a phenolic structure [(2-aminopropyl)-
(methylthio)phenol]; (iii) ?-hydroxylation of the side
chainto4-methylthionorephedrine
(methylthio)-phenyl]propan-1-ol) (Ewald et al., 2005).
The same metabolic pathways were observed in vivo in
mice (Carmo et al., 2002) and in vitro in primary hepa-
tocytes from man, monkey, dog, rabbit, rat, and mouse
(Carmo et al., 2004).
The enzymes involved in the metabolism of 4-MTA
arenotyetknownbutitisanticipatedthatthecytochrome
P450 isoenzymes (CYP) play a critical role in these
metabolic reactions. Of major importance is CYP2D6
that catalyses the ring hydroxylation, demethylenation,
and demethoxylation of amphetamines and derivatives
(Bach et al., 1999).
CYP2D6 activity shows a very high degree of
interindividual variability, which is primarily due to
genetic polymorphism that influences both enzyme
expression and function (Zanger et al., 2004). The
CYP2D6geneishighlypolymorphic,with58allelesand
morethan100distinctvariantslistedsofarattheCYPal-
lele nomenclature homepage http://www.imm.ki.se/
CYPalleles/. There is a marked interethnic variability
in the alleles frequencies among populations (Bertilsson
et al., 2002). CYP2D6 protein and enzymatic activity
is completely absent in less than 1% of Asians and up
to 10% of Caucasians as a result of the expression of
null alleles that encode a nonfunctional protein prod-
uct. These individuals are termed poor metabolizers
(Zanger et al., 2004). Among the extensive metabo-
lizers (EM) the enzyme activity is highly variable and
can be markedly reduced in intermediate metaboliz-
ers (IM; heterozygous for a normal and a deficient
allele). The frequency of the IM phenotype was esti-
mated to be around 10–15% of the European population
(Zangeretal.,2004).Attheotherextreme,theultrarapid
metabolizers (UM) phenotype can be caused by alle-
les carrying multiple gene copies (Ingelman-Sundberg,
2005).Thefrequenciesoftheseallelesaremarkedlyhigh
in Ethiopian and Saudi Arabians populations, and up to
5% of the Caucasians present this phenotype (Zanger et
al., 2004).
The aim of the present study was to determine if
the CYP2D6 genotype influences 4-MTA cytotoxicity.
We therefore tested 4-MTA cytotoxicity in a battery
of genetically engineered V79 cell lines expressing the
CYP2D6 allelic variants *1 (wild type), *2, *9 (reduced
(2-amino-1-[4-

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H. Carmo et al. / Toxicology 229 (2007) 236–244
Fig. 1. Proposed metabolic pathways for the phase I metabolism of 4-MTA in humans. These metabolic pathways include ?-hydroxylation of
the side chain into 4-methylthionorephedrine (I), ring hydroxylation into a phenolic metabolite (2-aminopropyl)-(methylthio)phenol (II), oxida-
tive deamination into a ketone metabolite 1-[4-(methylthio)phenyl]propan-2-one (III) followed by reduction into the corresponding alcohol
1-[4-(methylthio)-phenyl]propan-2-ol (IIIa), or degradation of the side chain into 4-methylthiobenzoic acid (IIIb).
activity alleles), and in a V79 cell line expressing
CYP3A4.
2. Materials and methods
2.1. Materials
Reagents for cell culture were obtained from Cambrex
Bio Science (Walkersville, MD, USA): Dulbecco’s modified
Eagle’s medium (4.5g/L glucose, with l-glutamine, without
sodium pyruvate; DMEM), phosphate-buffered saline (PBS),
trypsin(2.5%), penicillin
(10mg/mL) mixture, and foetal bovine serum (FBS). 4-MTA
(HCl salt) was synthesised by REQUIMTE/Toxicology
Department, Faculty of Pharmacy, University of Porto. All
other chemicals were purchased from Sigma-Aldrich (St.
Louis, Mo, USA) and were of the highest grade commercially
available.
(10,000U/mL)-streptomycin
2.2. Cell culture
Chinese hamster lung fibroblast V79 cell lines were
obtained from Prof. Johannes Doehmer and Dr. Niels Kreb-
sfaenger, GenPharmTox BioTech AG, Martinsried, and were
previously characterised for stable expression, activity, and
CYPcontent(Krebsfaengeretal.,2003).Atotalnumberofsix
celllinesweretested.Twoofthesecelllinesservedascontrols.
The parental cell line consisted of nontransfected cells. An
additional control was made by using a mock-transfected cell
line that carries the selective marker for neomycin resistance
(mockneo). Both control cell lines have no CYP-dependent
enzymatic activity. The transfected cell lines were desig-
nated according to the corresponding cDNA: CYP2D6*1,
CYP2D6*2, CYP2D6*9, and CYP3A4.
The cells were cultured in 75cm2flasks and the cell
cultures were kept at 37◦C in a humidified atmosphere con-
taining5%CO2/95%air.ThecellculturemediumwasDMEM

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H. Carmo et al. / Toxicology 229 (2007) 236–244
239
supplemented with 10% FBS and penicillin/streptomycin
(100U/100?g/mL). The cell cultures were routinely passaged
every 48h before reaching confluence. All the assays were
performed on at least three separate occasions, during differ-
entculturepassages,andeachtestsubstanceconcentrationwas
tested in triplicate in each experiment.
2.3. Colony formation assay
Allcelllineswereassayedbeforetheculturesreached100%
confluence. A cellular suspension was obtained by adding a
0.25% trypsin/1mM EDTA solution to the cell culture flasks
followed by 2min incubation at 37◦C and suspension of the
cellsincellculturemedium.Thecellswerecounted,givenonto
culture dishes at a density of 200cells/dish/9mL DMEM and
allowedtoattachfor4hat37◦C.Thesolutionsof4-MTAwere
freshlypreparedinPBSandweresterilefiltered.Foreachcon-
centrationtested(125–1000?M),a10×concentratedsolution
was prepared and 1mL of this solution was added to the cell
culture dishes 4h after plating the V79 cells. For the control
incubations, 1mL of PBS was added (solvent control). After
24h of exposure to the test substance, the cell culture medium
wasremovedandreplacedwith10mLoffreshDMEMandthe
cells were kept in culture for another 7days. Finally, the cell
colonieswerefixedwithanice-coldmixtureofethanol/acetone
(1:1),stainedwitha10%aqueoussolutionofGiemsablue,and
the number of colonies was counted. The number of colonies
expressedasapercentofthecorrespondingvalueofthesolvent
control incubations was used as a measure of cytotoxicity.
2.4. Statistical analysis
Resultsarepresentedasmean±S.E.M.Datawereobtained
from at least three representative experiments performed inde-
pendently. The means were compared by one-way analysis
of variance (ANOVA) followed by the Bonferroni’s multiple
comparison post hoc test, using GraphPad Prism (Graph-
Pad Software Inc., San Diego, CA, USA). Significance was
accepted at a p-value of less than 0.05. Details of the statistical
analyses are described in each figure legend.
3. Results
Toxicity of 4-MTA was tested in V79 cells trans-
fected with CYP2D6*1, CYP2D6*2, CYP2D6*9, and
CYP3A4 at concentrations of 125–1000?M. Interest-
ingly, clear differences in susceptibility were observed
between these cell lines. Cytotoxicity was increased in
the V79 cell line transfected with the human wild type
CYP2D6*1 enzyme (Fig 2C) compared to the parental
cell line (V79 parental; Fig. 2A) and to V79 cells trans-
fected with a neomycin resistance gene (V79 mockneo;
Fig. 2B). In V79 cells expressing CYP2D6*1, a signifi-
cantdecreaseinviabilitycomparedtothesolventcontrol
incubationwasalreadyobservedatthelowesttestedcon-
centration of 125?M 4-MTA with a 17% reduction of
the number of colonies formed when compared to the
solvent control incubations without 4-MTA. After incu-
bation with 250?M 4-MTA CYP2D6*1 caused a 21%
decrease in colony formation and at 500?M 4-MTA the
decrease was of 80% (Fig. 2C). In contrast, 250?M 4-
MTAdidnotcauseasignificantdecreaseinV79parental
and V79 mockneo colony formation.
When comparing the cytotoxicity of MTA in
V79 cells transfected with CYP2D6*1, CYP2D6*2,
CYP2D6*9, and CYP3A4 to the parental cell line (V79
parental) and to V79 cells transfected with a neomycin
resistance gene (V79 mockneo) after incubation with
500?M 4-MTA, only the V79 cell line transfected with
CYP2D6*1 caused a decrease in colony formation that
was significantly higher compared to the control cell
lines (47% for parental cells and 38% for mockneo
cells versus 80% for CYP2D6*1) (Fig. 3). CYP2D6*2
and CYP2D6*9 increased susceptibility to 4-MTA
with a decrease in colony formation of 60% and 70%,
respectively, but this increase was not statistically
different from the control cell lines (Fig. 3). In contrast
to CYP2D6, CYP3A4 did not significantly alter 4-MTA
toxicity compared to V79 parental and V79 mockneo
(Fig. 3). Generally, the highest 4-MTA toxicity was
observed in V79 cells expressing CYP2D6 wild type
(CYP2D6*1).
4. Discussion
Evidence of pharmacokinetic changes in amphe-
tamine derivatives due to pharmacogenetic-related defi-
ciency in CYP2D6 has been given both by in vitro
(Ramamoorthy et al., 2001; Ramamoorthy et al., 2002;
Tucker et al., 1994) and very recently by in vivo stud-
ies in humans (de la Torre et al., 2005). However, the
consequences of these pharmacokinetic changes on the
toxicityinducedbythesedrugsarestilltobedetermined.
In the present study we compared the cytotoxicity
of 4-MTA in V79 cells expressing wild-type CYP2D6
(CYP2D6*1) or the low activity alleles CYP2D6*2 and
CYP2D6*9.Ourresultsshowthattheexpressionofwild
type CYP2D6*1 greatly enhances the susceptibility to
the cytotoxic effects of 4-MTA compared to the parental
cells devoid of CYP-dependent enzymatic activity. Tox-
icity in V79 CYP2D6*1 was also higher compared to
the V79 cell lines expressing the low activity alleles
CYP2D6*2 and CYP2D6*9. In contrast to CYP2D6,
the CYP3A4 isoenzyme did not enhance 4-MTA
toxicity.
The V79-derived cell lines provide an in vitro system
with a high predictive value for humans in the testing of

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H. Carmo et al. / Toxicology 229 (2007) 236–244
Fig.2. 4-MTAcytotoxicitytowardstheV79celllinesexpressingdifferentvariantsofCYP2D6[parental;mockneo;CYP2D6*1(h2D61);CYP2D6*2
(h2D62); CYP2D6*9 (h2D69); and CYP3A4 (h3A4)]. Toxicity was determined by the colony formation assay (A–F). All cell lines were exposed to
0.125,0.25,0.5,and1mM4-MTAfor24hunderstandardcellcultureconditions.Thenumberofcoloniesexpressedaspercentofthecorresponding
value of the solvent control incubations was used as a measure of cytotoxicity (% solvent control). Data are mean±S.E.M. and were obtained
from three independent experiments. The means were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni’s multiple
comparison post hoc test. **p<0.01 and ***p<0.001 compared to solvent control.
CYP2D6 polymorphisms. These cells have been exten-
sively characterised for stable expression of mRNA, for
enzymeactivityusingthemodelsubstratebufuralol,and
for their CYP content (Krebsfaenger et al., 2003). They
were found to express activities close to those observed
in native tissue. The measurement of the CYP content
showed some variation which could also account for
the enzyme kinetic differences between the cell lines.
However, this cell model was fully validated with the
hydroxylationoftheCYP2D6modelsubstratebufuralol

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241
Fig. 3. Each of the V79 cell lines transfected with the variant CYP
enzymes [CYP2D6*1 (h2D61); CYP2D6*2 (h2D62); CYP2D6*9
(h2D69); and CYP3A4 (h3A4)] were compared to the parental and
mockneo control cell lines after incubation with 500?M 4-MTA. The
number of colonies expressed as percent of the corresponding value
of the solvent control incubations was used as a measure of cytotox-
icity (% solvent control). Data are mean±S.E.M. and were obtained
from three independent experiments. The means were compared by
one-way analysis of variance (ANOVA) followed by the Bonferroni’s
multiple comparison post hoc test. **p<0.01 compared to mockneo
control cell line.&p<0.05 compared to parental control cell line.
and also with the selective estrogen receptor tamoxifen
(Krebsfaenger et al., 2003).
Interindividualdifferences
amphetamine-related designer drugs toxicity have been
noticed(Carmoetal.,2005;Coladoetal.,1995;deLetter
etal.,2004;Henryetal.,1992;JonesandSimpson,1999;
Malpass et al., 1999; O’Donohoe et al., 1998; Tucker et
al.,1994).Inapreviousinvitrostudylargedifferencesin
susceptibility to 4-MTA cytotoxicity were found using
hepatocytes from three human donors (Carmo et al.,
2004).Thisraisedthehypothesisthattheinterindividual
differences in metabolic capacity of these hepatocytes
might be responsible for the observed differences in
4-MTA cytotoxicity. It has been increasingly acknowl-
edged that the toxic effects of the amphetamine designer
drugs are not only related to high plasma levels of
the drug (Greene et al., 2003) but also to metabolism.
In fact, several studies have implicated the oxidative
metabolism catalysed by CYP2D6 on the neurotoxic
(Capelaetal.,2006;Eastonetal.,2003;Gollamudietal.,
1989; Jones et al., 2005), hepatotoxic (Carvalho et al.,
2004a,b), cardiotoxic (Carvalho et al., 2004c), nephro-
toxic (Carvalho et al., 2002), hyponatraemia (Forsling
et al., 2002; Hartung et al., 2002), and hyperthermic
(Escobedo et al., 2005) effects associated with these
drugs.
CYP2D6 shows a very high degree of interindivid-
ual variability, which is primarily due to the extensive
genetic polymorphism that influences both enzyme
expression and function (Zanger et al., 2004). Concern
in susceptibilityto
has been raised that individuals who lack functional
CYP2D6 alleles may be at risk of acute amphetamine
intoxications, such as increased heart rate and hyper-
tension due to the higher plasma levels of the drugs.
Additionally, the combination of these drugs with
CYP2D6 inhibitors may give rise to pharmacokinetic
interactions of toxicological relevance. Such is the
case of dextromethorphan which is sometimes found
in “ecstasy” pills (Baggott et al., 2000), selective sero-
tonin reuptake inhibitors (SSRIs) such as fluoxetine and
paroxetine(Seguraetal.,2005),MAOinhibitorssuchas
moclobemide(Smilksteinetal.,1987;Vuorietal.,2003),
and ritonavir that was implicated in a fatal intoxication
with MDMA (Henry and Hill, 1998).
The hypothesis that poor metabolizers may be at
increased risk of acute intoxication comes from the
assumptionthattheseverityofthetoxiceffectscorrelates
withtheserumconcentrationsofthedrugs(Greeneetal.,
2003).However,thisisnotcorroboratedbyseveralstud-
ies that have shown that there is a lack of dose-response
relationship after amphetamine administration (Asghar
et al., 2003; Farr´ e et al., 2004; Mas et al., 1999). This
could explain why, at least in some individuals, toxic
reactions may be unrelated to the amount taken, indicat-
ing that other factors beyond plasma concentration play
a role in the induction of acute toxic effects.
The concern regarding the influence of the different
CYP2D6 genotypes on the susceptibility to the toxic
effects of the amphetamines prompted the investigation
of a possible association between the poor metabolizer
genotypeforCYP2D6andtheoccurrenceofacuteintox-
ication. Such an association was not found in any of the
studies reported so far thus indicating that the metabolic
impairment is not responsible for higher susceptibility
to toxicity (Gilhooly and Daly, 2002; O’Donohoe et al.,
1998; Schwab et al., 1999). In the light of our findings
we hypothesise that individuals with higher metabolic
capacity for CYP2D6 may in fact be at a higher risk of
intoxication when compared to the poor metabolizers. It
would be of great interest to investigate whether the UM
phenotype(upto5%oftheCaucasians)characterizedby
multiple CYP2D6 gene copies is over represented in the
cases of 4-MTA and other amphetamine designer drug-
related intoxications. According to our results this may
be more relevant than the genotyping of the most com-
mon deficient alleles that has already been performed in
previous studies (Gilhooly and Daly, 2002; O’Donohoe
et al., 1998; Schwab et al., 1999).
Recently, we have shown that toxicity of 3,4-methy-
lenedioxymethamphetamine
increased in cells expressing CYP2D6*1 compared to
the parental cells devoid of CYP-dependent enzymatic
(MDMA)wasclearly

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H. Carmo et al. / Toxicology 229 (2007) 236–244
activity (Carmo et al., 2006). Formation of the highly
toxic MDMA metabolite N-methyl-?-methyldopamine
(N-Me-?-MeDA) by CYP2D6*1 was responsible for
increased toxicity. Whether a similar CYP2D6 depen-
dent metabolic pathway also contributes to 4-MTA
toxicity still has to be determined.
The concentrations of 4-MTA used in the present
study are in the range of concentrations used in sev-
eral in vitro studies (Capela et al., 2006; Carvalho et
al., 2004b,c; Simantov and Tauber, 1997; Stumm et al.,
1999). They are higher than blood concentrations found
inhumanabusers.Inthereportedfatalintoxicationswith
4-MTA interindividual variations in blood levels could
be observed (between 7.7 and 29.6?M) (Decaestecker
et al., 2001; Elliott, 2000; EMCDDA, 1999; Poortman
and Lock, 1999). However, the tissue concentrations
can be substantially higher. Post-mortem liver and
brain concentrations of 170 and 201.6?M, respec-
tively, were reported in a fatal intoxication with 4-MTA
(Decaestecker et al., 2001). Moreover, autopsy findings
in other amphetamine-related intoxications have shown
that the tissue levels of the drug in the liver can be up
to 18 times higher than blood concentrations (de Letter
et al., 2006) and 30 times higher in the brain (Garcia-
Repetto et al., 2003). It should also be noted that these
concentrations found at autopsy are probably lower than
the peak concentrations that are expected to occur after
drug intake, especially in the cases where the victims
are submitted to emergency-care treatments to control
the intoxications.
In conclusion, we have shown that CYP2D6*1 medi-
ates higher levels of 4-MTA toxicity than CYP2D6*2
and CYP2D6*9 as well as CYP3A4. The data presented
in this study provide evidence that the pharmacoki-
netic differences in amphetamine metabolism due to the
genetic polymorphism of CYP2D6 are of major impor-
tance for the expression of the cytotoxic effects of these
drugs.
Acknowledgments
ThisworkwassupportedbyFundac ¸˜ aoParaaCiˆ encia
e a Tecnologia (POCI/SAU-FCF/57187/2004), by
REQUIMTEAssociatedLaboratory,byaCRUP/DAAD
grant (reference A-5/04) and by the German Federal
Ministry of Education and Research (BMBF), funding
priority HepatoSys, Systems Biology of the Hepatocyte.
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