Pharmacogenetics and forensic toxicology

Frank Musshoff
a,
*, Ulrike M. Stamer
b
, Burkhard Madea
a
a
Institute of Forensic Medicine, Stiftsplatz 12, 53111 Bonn, Germany
b
Department of Anaesthesiology and Pain Therapy, Inselspital, University Hospital Bern, Freiburgstr., 3010 Bern, Switzerland
1. Introduction
As reviewed by Eichelbaum et al. [1], most patient populations
showlarge inter-individual variabilitywithrespect todrugresponse
and toxicity. Even when administered at standard doses, a
substantial proportion of patients fail to respond, respond only
partially, or experience adverse drug reactions (ADRs) for all major
classes of drugs (e.g., opioids, tricyclic antidepressants, serotonin
reuptake inhibitors, ACE inhibitors, b-adrenoreceptor agonists,
statins, etc.). For two individuals of the same weight on the same
dose, drug concentrations in plasma can vary substantially. This
variationcanbeof physiological, pathophysiological, environmental
or genetic origin. In general, potential risk factors for ADRs include a
patients age, sex, co-morbidities, co-medication, organ dysfunction
(especially of liver and kidneys), diet, as well as some lifestyle
variables, such as smoking habits and alcohol intake (Fig. 1).
However, a drugs absorption, distribution, metabolism and inter-
actions withits target oftenaredeterminedbygeneticdifferences. In
general, geneticfactors are estimatedtoaccount for 1530%of inter-
individual differences in drug metabolism and response, but for
certain drugs this can be as high as 95% [35].
Pharmacokinetic and/or pharmacodynamic variations can
appear at the level of
drug metabolizing enzymes;
drug transporters;
drug targets;
other biomarker genes.
Pharmacogenetics or toxicogenetics can also be relevant in
forensic toxicology [6,7]. For this reason, appropriate aspects are
presented, together with some examples from daily routines.
2. Reasons for pharmacokinetic variations
2.1. Drug metabolism
The hepatic cytochrome P450 system is responsible for the rst
phase of the metabolismand elimination of numerous endogenous
and exogenous molecules and ingested chemicals. The P450
enzymes convert these substances into electrophilic intermedi-
ates, which are then conjugated by phase II enzymes (e.g., UDP
glucuronosyltransferases; N-acetyltransferases) to form hydro-
philic derivatives that can be excreted [8]. The P450 superfamily
comprises several subfamilies that are designated by letter
according to the system of nomenclature recommended by an
international committee [911] and data are continuously
upgraded [12]. Although humans probably have at least 60 unique
P450 genes within several subfamilies [10], only a subset (e.g.
CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP2E1, and
CYP3A4) is responsible for metabolism of the vast majority of
prescribed and over-the-counter drugs [1327]. Table 1 presents a
list of selected drugs that are metabolized by specic cytochrome
P450 isoforms.
Forensic Science International 203 (2010) 5362
A R T I C L E I N F O
Article history:
Available online 9 September 2010
Keywords:
Pharmacogenetics
Forensic toxicology
Polymorphism
P450
CYP2D6
Tramadol
Codeine
Antidepressants
A B S T R A C T
Large inter-individual variability in drug response and toxicity, as well as in drug concentrations after
application of the same dosage, can be of genetic, physiological, pathophysiological, or environmental
origin. Absorption, distribution and metabolism of a drug and interactions with its target often are
determined by genetic differences. Pharmacokinetic and pharmacodynamic variations can appear at the
level of drug metabolizing enzymes (e.g., the cytochrome P450 system), drug transporters, drug targets
or other biomarker genes. Pharmacogenetics or toxicogenetics can therefore be relevant in forensic
toxicology. This review presents relevant aspects together with some examples from daily routines.
2010 Published by Elsevier Ireland Ltd.

This paper is part of the special issue entitled: Molecular Pathology in Forensic
Medicine, Guest-edited by Burkhard Madea and Pekka Saukko.
* Corresponding author. Tel.: +49 0228 73 83 15; fax: +49 0228 73 83 68.
E-mail address: f.musshoff@uni-bonn.de (F. Musshoff).
Contents lists available at ScienceDirect
Forensic Science International
j our nal homepage: www. el sevi er . com/ l ocat e/ f or sci i nt
0379-0738/$ see front matter 2010 Published by Elsevier Ireland Ltd.
doi:10.1016/j.forsciint.2010.07.011
Because of genetic polymorphism of the CYP P450 enzymes,
populations could be classied into three major phenotypes [28]:
the ultra-rapid metabolizers (UM), with more than two active
genes encoding a certain P450;
the extensive metabolizers (EM), carrying two functional genes;
the poor metabolizers (PM), lacking functional enzymes due to
defective or deleted genes;
heterozygous individuals, carrying one functional and one
defective PM associated allele
and intermediate metabolizers (IM) carrying alleles partially
decreasing enzyme activity. These genotypes have reduced but
not absent CYP activity.
Subjects with PM associated variants are at increased risk of
suffering from adverse side effects due to drug overdose or of
experiencing therapeutic failure due to poor metabolism of a
prodrug to the active metabolite (Fig. 2). In contrast, UMs have
signicantly increased enzyme activity, which can result in

Fig. 1. Variables determining response to a drug. Modied from [2].

Table 1
Selection of substrates, inhibitors and inducers of specic cytochrome P450 isoforms.
Enzyme Substrates Inhibitors Inducers
CYP1A2 Antidepressants: amitriptyline, clomipramine, duloxetine, uvoxamine, imipramine,
maprotiline, mirtazapine
Antipsychotics: clozapine, haloperidol, olanzapine, promazine, thioridazine, zotepine
Others: aminopyrine, amiodarone, caffeine, melatonin, paracetamol, phenacetin, propafenone, propanolol,
tacrine, theophylline, verapamil, R-warfarin, zolpidem
Ciprooxacin
Enoxacin
Fluvoxamine
Barbiturates
Carbamazepine
Phenytoin
Rifampicin
Smoking
CYP2B6 Others: bupropion, cyclophosphamide, desmethylseledeline, methadone Phenobarbital
Rifampin
CYP2C9 NSAIDs: diclofenac, ibuprofen, naproxen, piroxicam
Others: losartan, phenobarbital, phenytoin, tolbutamide, valproic acid, valsartan, S-warfarin
Fluconazole
Fluoxetine
Fluvoxamine
Sulfaphenazole
Barbiturates
Carbamazepine
Phenytoin
Rifampicin
CYP2C19 Antidepressants: amitriptyline, citalopram, clomipramine, escitalopram, imipramine
Others: diazepam, lansoprazole, omeprazole, phenytoin, proguanil, propanolol
Fluvoxamine
Omeprazole
Ticlopidine
Barbiturates
Carbamazepine
Phenytoin
Rifampicin
CYP2D6 Antiarrhytmics: encainide, ecainide, propafenone
Antidepressants: amitriptyline, citalopram, clomipramine, desipramine, escitalopram,
uoxetine, uvoxamine, imipramine, mirtazapine, nortriptyline, paroxetine, venlafaxine
Antipsychotics: aripiprazole, clozapine, haloperidol, olanzapine, perphenazine,
risperidone, sertindole, thioridazine
b-Blockers: alpenolol, bufuralol, metoprolol, propanolol, timolol
Others: atomoxetine, codeine, donepezil, galantamine, phenformin, tramadol
Bupropion
Duloxetin
Fluoxetine
Paroxetine
Quinidine
Thioridazine
CYP3A4 Antidepressants: amitriptyline, citalopram, clomipramine, escitalopram, imipramine,
mirtazapine, nefazodone, sertraline, venlafaxine
Antipsychotics: aripiprazole, clozapine, haloperidol, pimozide, quetiapine, risperidone,
sertindole, ziprasidone
Benzodiazepines: alprazolam, clonazepam, diazepam, midazolam, triazolam
Calcium channel antagonists: diltiazem, felodipine, nifedipine, verapamil
Statins: atorvastatin, lovastatin, pravastatin, simvastatin
Immunosuppressants: cyclosporine, sirolimus, tacrolimus
Others: amiodarone, astemizole, carbamazepine, cisapride, clarithromycin,
donepezil, erythromycin, ethinyl estradiol, galantamine, levonorgestrel,
methadone, quinidine, ritonavir, tamoxifen, terfenadine
Erythromycin
Grapefruit juice
Iraconazole
Ketoconazole
Nefazodone
Ritonavir
Troleandomycin
Barbiturates
Carbamazepine
Phenytoin
Rifampicin
Ritonavir
F. Musshoff et al. / Forensic Science International 203 (2010) 5362 54
subtherapeutic blood levels of the enzyme substrate. In case of a
prodrug the metabolism to the active metabolite is increased.
Functional CYP polymorphisms consist of single nucleotide
polymorphisms (SNPs) accounting for the majority of genetic
variants, gene deletions, gene duplications. Deleterious genetic
variants that create inactive gene products might be; e.g., small
insertions and deletions causing frame shift mutations. [28].
Mutations or polymorphisms in introns can create altered splice
sites, whilebaseexchanges inexonscanresult inaminoacidchanges
with possible effects on substrate specicity. The copy number
variation, where multiple functional gene copies of one allele can
result inincreaseddrugmetabolism, canalsobeanimportant aspect
in genetically determined absence of drug response or in case of a
prodrug, drug-related adverse events and fatalities.
2.2. Drug transporters
Transporters and transporter-related proteins play a major role
in drug absorption, disposition, toxicity and efcacy and signicant
transporter-mediated drug interactions can occur [29]. These types
of interactions can be inhibitory, inductive, or both, and may
involve inux or efux transporters. They are of interest if: (1) the
elimination of the affected drug or the distribution into a target
tissue is mediated primarily by the transporter and (2) the
interaction results in the concentration of the affected drug at the
site of action [30,31].
For example, the P-glycoprotein (P-gp) efux pump belongs to
the ATP-binding cassette (ABC) family transporters and is encoded
by the MDR1 gene (or ABCB1). In humans, P-gp is present in several
tissues that are important for drug absorption, distribution and
elimination (Fig. 3). Due to the broad substrate specicity of P-gp,
inhibitors or inducers of P-gp may produce signicant drugdrug
interactions.
Another important group are the organic anion (protein)
transporters (OATPs or OATs) encoded by the solute carrier gene
family (SLC). These Na
+
-independent transporters partly rely on
co-transport and are responsible for the transport of a wide range
of endogenous compounds as well as drugs.
Organic cationtransporter (OCT) interactions, as well as peptide
transporter (PEPT) and nucleoside transporter (NT) interactions,
have also been described.
The TransportDB website currently lists as many as 1.022
transporters for Homo sapiens alone [32]. For example, the so-called
Pharmacogenomics of Membrane Transporters (PMT) project is at
the crossroads of pharmacogenomics and transporter biology.
Recently, allele frequencies and ndings regarding functional

Fig. 2. Pharmacological effects depending on metabolism of an active drug (A) or

prodrug (B). Genotypes determine enzyme activity and metabolism of a drug, thus
causing increased (overdose/side effects), normal, or lacking pharmacological
effects of a drug. Modied from [2].

Fig. 3. In humans, P-gp is present in several tissues important for drug absorption, distribution and elimination, such as the apical membrane of intestinal epithelial cells, the
canalicular membrane of the hepatocytes, the capillary endothelial cells of the brain, the apical membrane of the placental syncytiotrophoblasts and the apical membrane of
the renal proximal tubular cells. In these tissues P-gp functions as an efux pump are involved in the entry of substances into these tissues. Reprinted from [30] with
permission from Elsevier.
F. Musshoff et al. / Forensic Science International 203 (2010) 5362 55
variants in drug transporter systems were summarized by Kroetz
et al. [33].
2.3. Drug targets
Pharmaceutical agents generally exert their effects by binding
to and subsequently modulating the activity of specic protein,
nucleic acid or other molecular (such as membrane) targets, such
as the b
2
-adrenoceptors, insulin receptors, angiotensin-converting
enzyme and others. Factors such as drug interactions and
especially polymorphisms in genes encoding these targets may
inuence the sensitivity to selected drugs. These polymorphisms
are considered to be signicant in cases where inter-individual
variations in drug plasma concentrations are minimal, but where
major pharmacodynamic differences can be observed [6]. Infor-
mation about therapeutic targets are given by several databases
such as Drugbank [34], Potential Drug Target Database (PDTD)
[35], or Therapeutic Target Database (TTD) [36].
Pathway databases are alsohelpful for the understanding of drug
interactions; e.g., the well-knowndruginteractiontable of Flockhart
[37], the TherapeuticallyRelevant MultiplePathways (TRMP) [38] or
the Small Molecule Pathway Database (SMPDB) [39] with informa-
tion on the relevant organs, organelles, subcellular compartments,
protein cofactors, protein locations, metabolite locations, etc.
According to the authors, the concept of metabolic pathway
mapping has actually proven to be so effective that is has been
extended to describe protein signalling, proteinDNA interactions
and many other molecular biological phenomena.
2.4. Other biomarkers
According to the National Institutes of Health working group, a
biomarker is a characteristic that is objectively measured as an
indicator of normal biological processes, pathogenic processes, or a
pharmacological response to a therapeutic intervention [40]. In the
past, physiological parameters such as body temperature, blood
pressure, colour of urine, heart beat rate, respiratory sounds or a lot
of parameters deriving from clinical chemistry such as serum
creatinine, urinary glucose or enzymes in blood or urine have been
considered as potential biomarkers. Recently, Marrer and Dieterle
[41] reviewed biomarkers for kidney safety, including serum
cystatin C, expression markers KIM-1 (kidney injury molecule-1)
and lipocalin-2 (NGAL), urinary b2-microglobulin for the moni-
toring of tubular re-absorption or total protein for the glomerular
ltration membrane. Biomarkers for liver safety (a-gluthathione-
S-transferase (GST-a), g-glutamyl-transpeptidase (GGT), paraox-
ygenase-1 (PON-1), purine nucleotide phosphorylase (PNP),
malate dehydrogenase (MDH)), cardiac safety (cardiac troponin
1 and T (cTn-1 and cTn-T)), natiuretic peptides (B-type natiuretic
peptide (BNP)), and vascular safety (Willebrand factor (vWF), vWF
propeptide (vWFpp), caveolin-1 (Cav-1)) have also been presented.
However, all of these markers are more useful in drug safety
assessment during or in drug development.
Today, the term biomarker increasingly refers to molecular and
cellular markers joined with the development of modern scientic
elds like proteomics, metabolomics, pharmacogenomics or better
toxicogenomics. Reactive molecules produce DNA adducts, protein
adducts andmetabolites andrepresent biomarkers of exposure [42].
Many DNA adducts can result in mutations, while irreversible
changes in the DNA structure can alter the genetic information
content. Mutations represent biomarkers of effect. Microarray
technologies can move biomarker discovery forward and result in
genomic/genetictests, proteinassaysor other analytical tests[43,44].
The application of mass spectrometric procedures is useful for such
purposes [45].
3. Ethnic variations in drug dispositions
A drug metabolism phenotype is partly determined by the
inheritance of various alleles, which exhibit variations among
different ethnic groups. For example, the frequency of the CYP2D6
PM phenotype is much higher in population groups of Western
Caucasian origin (510%) than in populations fromthe Far East or in
Asian ethnic groups (02%) [4650]. Major human polymorphic
variant CYP2D6 alleles and their global distribution are described in
Table 2. The frequency of PMs with the CYP2C19 allele is lower in
Western Caucasians (24%) compared to the frequencies observed
amongOrientals (1525%), where it reaches as highas 6070%inthe
Pacic islands [5052]. Recently Sistonen et al. [20] described
altered activity variants of CYP2C9, CYP2C19, and CYP2D6 occurring
globally in all geographic regions and reported these to reach
extremely high frequencies in some populations. Each of the CYP
genes studied showed a distinct geographic pattern of variation and
population substructure that can strongly affect variations seen in
pharmacogenetic loci. However, according to this study, several
geographic regions are still poorly characterized. Global heteroge-
neity and ethnic differences have also been reported with respect to
the frequency of variant alleles of the genes expressing many other
drug metabolizing enzymes [50,5357] as well as the gene
expressing P-gp [58,59]. Other ethnic differences also exist in the
mutations of a number of drug targets, for example, those
concerningtheb-adrenoreceptor [60] or theserotoninreceptor [61].
Consequently, ethnic variations in drug metabolism pheno-
types as well as differences in drug transporters or drug targets
should be taken into consideration in both clinical as well as
forensic cases.
4. Applications in forensic toxicology
Interpretation of forensic-toxicological results is a task of great
importance that demands knowledge of many different aspects of
analytical toxicology, as well as pharmacokinetics and pharmaco-
dynamics [62]. Especially in post-mortem toxicology, further
aspects such as site- and time-related differences in post-mortem
drug concentrations have to be considered, which complicates
extrapolation from post-mortem to ante-mortem concentrations
Table 2
Major human polymorphic variant CYP2D6 alleles and their global distribution [1].
Major variant allele
a
Mutation Consequence Allele frequency (%)
Caucasians Asians Black
Africans
Ethiopians and
Saudi Arabians
CYP2D6*2xn Gene duplication/multiduplication Increased enzyme activity 15 02 2 1016
CYP2D6*4 Defective splicing Inactive enzyme 1221 1 2 14
CYP2D6*5 Gene deletion No enzyme 27 6 4 13
CYP2D6*10 P34S, S486T Unstable enzyme 12 51 6 39
CYP2D6*17 T107I, R296C, S486T Altered afnity for substrates 0 0 2035 39
a
All variant alleles are listed by the Human CYP Allele Nomenclature Committee at http://www.imm.ki.se/cypalleles/cyp2d6.
F. Musshoff et al. / Forensic Science International 203 (2010) 5362 56
[6367]. In daily routine, concentrations of drugs have to be
compared with the same kind of reference values for both
therapeutic and toxic levels. Ratios of parent drug to metabolite
concentrations (P/M ratio) can be helpful to decide whether an
intake was acute (high ratio) or the result of a chronic use.
However, other variables also inuence the P/M ratio, including
interactions between different drugs and genetic variations
described above. For these reasons, genotyping has become more
common in forensic toxicology. Holmgren and Ahlner [62]
developed a scheme for determining the appropriateness of
genotyping in several clinical or forensic settings (Table 3). Some
case studies and case reports relevant to forensic toxicology are
presented here. Although investigations and reports on genetically
determined fatalities are still sparse up to date, modern
biomarkers are expected to become a useful tool for forensic
medicine in the future.
4.1. Opioids
The CYP2D6 system, in particular, is responsible for the
metabolismof a number of opioid drugs such as codeine, tramadol,
dihydrocodeine, oxycodone, hydrocodone, ethylmorphine, and at
least part of methadone. The CYP2D6 PM phenotype affects opioid
analgesics in different ways [68]: it reduces analgesic effects of
codeine, decreases the clearance of methadone and reduces the
efcacy of tramadol.
4.1.1. Tramadol
The weak racemic opioid tramadol is metabolized by hepatic
CYP2D6 to O-demethyltramadol (ODT) (Fig. 4). (+)-ODT has been
shown to have an afnity for m-opioid receptors that is
approximately 200 times greater than that of the parent drug.
Thus, (+)-ODT is largely responsible for opioid receptor mediated
Table 3
Situations in which genotyping could give additional information. Modied according to Holmgren and Ahlner [62].
Analytical ndings or other information Factors to determine
Is a polymorphic
enzyme involved?
Interactions with
other drugs?
Perform
genotyping?
High ratio between parent drug and metabolite: accidental death or suicide? Yes Yes Consider
No Yes
No No
Abnormal high or low ratios between parent drug and metabolite Yes Yes Consider
No Consider
No No
Adverse drug reactions in anamnesis Yes Yes Consider
No Consider
No No
High ratio between parent drug and metabolite: intake according to prescription was claimed Yes Yes Consider
No Yes
No No

Fig. 4. Metabolic pathway of tramadol involving polymorphic CYP enzymes.

F. Musshoff et al. / Forensic Science International 203 (2010) 5362 57
analgesia, whereas (+)- and ()-tramadol contribute to analgesia
by inhibition of serotonin and noradrenaline reuptake.
Recently, the inuence of the CYP2D6 genotype and CYP2D6
inhibitors was investigated with respect to enantiomeric plasma
levels of tramadol and ODT as well as response to tramadol [69].
Concentrations of (+)-ODT differed in the four genotype groups
(PMs < IMs < EMs < UMs) and co-medication with CYP2D6 inhibi-
torsdecreased(+)-ODTconcentrations. InPMs, non-responserates to
tramadol treatment were increased 4-fold compared with the other
genotypes, allowing the conclusion that the CYP2D6 genotype
determines ODT concentrations and inuences efcacy of tramadol
treatment. Pharmacokinetic differences betweenEMs andUMs were
alsomeasuredby Kirchheiner et al. [70]; UMs were more sensitive to
tramadol than were EMs. According to the authors, tramadol may
frequently cause adverse effects in southern European and Northern
African populations that have a high proportion of UMs.
An opioid-related respiratory depression in a patient receiving
tramadol was reported by Stamer et al. [71]. Complete recovery
occurred after naloxone administration, thus conrming opioid
intoxication. Analysis of the patients genotype revealed a CYP2D6
gene duplication resulting in ultra-rapid metabolism of tramadol to
its active metabolite (+)-ODT. A concomitant renal impairment
resulting in decreased metabolite clearance further enhanced opioid
toxicity.
In 33 autopsy cases, Levo et al. [72] analysed both, the CYP2D6
genotype and the concentrations of tramadol and its metabolites O-
and N-demethyltramadol. They found a correlation between the
number of functional CYP2D6 alleles and the ratios of tramadol to
ODTas well of tramadol toN-demethyltramadol. Thenumber of UMs
was overrepresented in these fatalities. There were two important
ndings of this study: (1) genetic variation in drug metabolizing
enzymes can be analysed in post-mortem blood, and (2) genetic
variation correlates well with the parent drug to metabolite ratios.
4.1.2. Codeine
Comparabletotramadol, O-demethylationof theprodrugcodeine
toitsmetabolitemorphineis essential for itsopioidactivityand, thus,
the CYP2D6 genotype specically inuences the efcacy and side
effects (Fig. 5). Theanalgesic effect of codeine has beenreportedtobe
substantially reduced in subjects found to be PMs [68,7375]. In the
following some case reports concerning codeine are presented.
A 72-year-old man developed life-threatening respiratory
depression after a 3-day treatment with codeine (3 25 mg/
day). The adverse events were mediated by: (1) high concentra-
tions of morphine owing to an UM genotype; (2) transient
reduction in renal function with reduced clearance; and (3) co-
medication with clarithromycin and voriconazole that blocked
CYP3A4 as alternative pathway for codeine metabolism [76].
A breast-fed neonate whose mother received codeine 30 mg/
day died on day 13 of morphine poisoning. The mother had an UM
genotype and, thus, high amounts of morphine were formed from
codeine, which then were transferred to the baby [77].
A 29-month-old previously healthy child experienced apnea
resulting in brain injury following a dose of acetaminophen and
codeine 2 days after an uneventful anaesthetic for tonsillectomy. A
genetic polymorphism leading to ultra-rapid metabolism of
codeine into morphine resulted in narcosis and apnea [78].
However, CYP2D6 genotyping is not only performed to prove the
responsibility of genetic polymorphism for side effects. It is also
important to prove an(accidental) intoxication; e.g., by dosage error
whenalternatively a UMgenotype could be seenas responsible for a
fatality [79,80].
4.1.3. Dihydrocodeine
Similarly to codeine, dihydrocodeine is converted by CYP2D6 to
dihydromorphine, which has activity comparable to that of
morphine. EMs have been demonstrated to have 7-fold higher
dihydromorphine concentrations compared with PMs [81]. Quini-
dine-induced poor metabolism produced a 3- to 4-fold decrease in
dihydromorphine plasma concentrations [82].
4.1.4. Oxycodone
Oxycodone, a semi-synthetic opioid with m-receptor agonist-
mediated effects, is metabolized to its active metabolite oxymor-

Fig. 5. Codeine is metabolized into norcodeine by CYP3A4 and into codeine-6-glucuronide by glucuronidation usually to 80%, and conversion to morphine via CYP2D6
represents only 10% of codeine clearance. Morphine is further metabolized to morphine-6-glucuronide and morphine-3-glucuronide followed by renal elimination. Ultra-
rapid CYP2D6 metabolism perhaps with inhibition of CYP3A4 (co-medication) and acute renal failure can lead to unexpected morphine concentrations.
F. Musshoff et al. / Forensic Science International 203 (2010) 5362 58
phone by O-demethylation via the polymorphic CYP2D6. In a
recent clinical study, the mean oxymorphone/oxycodone ratios
were reported as 0.0031 and 0.00081 in EMs and PMs, respectively.
According to these results, the oxymorphone formation depends
on CYP2D6, but no differences were found in the post-operative
analgesic effect of intravenous oxycodone between the two
CYP2D6 genotypes [83]. By assessing the prevalence of CYP2D6
polymorphisms and covariables, oxycodone fatalities were also
hypothesised to be partially due to poor drug metabolism caused
by CYP2D6 variant alleles. A retrospective analysis of 15
oxycodone cases was followed by genotyping and chemical-
toxicological analysis. Genotyping provided a more denitive
interpretation of oxycodone toxicity in four cases. Therefore,
pharmacogenomics was proposed to serve as an adjunct in the
determination of the cause and manner of death in forensic
toxicology and a pharmacogenomic algorithm for genotyping has
been proposed [84]. However, as also described by others [85], the
PM genotype was rare and drugdrug interactions in EMs
constituted a more frequent and important nding.
4.1.5. Ethylmorphine
In a fatality of a 10-month-old boy ethylmorphine which is a
common ingredient of antitussive preparations, was administered.
Ethylmorphine is partially metabolized to morphine by CYP2D6.
Genotyping revealed an EMgenotype, signifying a normal capacity
for metabolizing ethylmorphine to morphine [86]. Autopsy report
concluded that death was associated with opioid-induced sedation
and respiratory depression, and a respiratory infection combined
with sleeping position that could have impeded breathing.
4.1.6. Methadone
Methadone is partially metabolized by CYP2D6 and methadone
toxicity was assumed to be partially due to CYP2D6*3, *4, and *5
variant alleles, resulting in poor drug metabolism. However, in a
retrospective analysis performed on covariables and risk factors of
21 methadone cases, CYP2D6 mutations were not shown as yet to
be directly associated with methadone toxicity [87].
Otherwise, methadone is mostly given as a racemic mixture,
but CYP2B6 metabolism is stereo-selective toward the (S)-
enantiomer. (R)-methadone mediates its narcotic effect by
activating the m-opioid receptor, whereas (S)-methadone inhibits
the cardiac potassium channel, contributing to the cardiac side
effects of methadone treatment. Decreased CYP2B6 activity was
found to be associated with high plasma concentrations of (S)- but
not (R)-methadone and with a greater risk of cardiac side effects
and death [88].
4.1.7. Fentanyl
The opioid fentanyl is metabolized via CYP3A4 to norfentanyl,
however, CYP3A5 is also a catalyst of fentanyl oxidation [89]. The
majority of the Caucasian population is carrier of the *3 allele, thus
resulting in lowered CYP3A5 activity. In an examination of
fentanyl-related deaths, homozygous CYP3A5*3 individuals
showed impaired metabolism of fentanyl if they additionally
carried the CYP3A4*1B, especially the homozygous genotype [90].
The authors concluded that genotyping of CYP3A4*1B and
CYP3A5*3 may serve as molecular autopsy.
4.2. Ecstasy
3,4-Methylendioxymethamphtamine (MDMA), the main ingre-
dient of ecstasy, is metabolized via CYP2D6 and PMs have been
suggested to possibly be at greater risk of becoming intoxicated by
this drug. However, in two small published studies, no PM was
found among individuals who died after taking MDMA [91,92].
Nonetheless, caution has to be taken, as misclassication of the
genotype might have been possible due to the number of SNPs
investigated. In addition to CYP2D6, several other P450 isoen-
zymes have the capacity to contribute to the oxidative metabolism
of MDMAs [93,94].
4.3. Benzodiazepines
In some cases, subjects who have taken prescribed drugs
according to a physicians directions cannot be prosecuted unless it
is proven that they had overdosed the medication. For example,
benzodiazepines, especially diazepam, commonly found in
drugged drivers, are metabolized via CYP2C19. A high P/M ratio
between diazepam and desmethyldiazepam indicates an acute
intake. Genotyping may be necessary in specic cases, because a
PM who has not deliberately overdosed will also show a high P/M
ratio [62].
4.4. Antidepressants and antiepileptics
A 9-year-old boy was treated with methylphenidate, clonidine,
and uoxetine, and over a 10-month period signs and symptoms
suggestive for metabolic toxicity were observed. Finally, he
suffered a status epilepticus and cardiac arrest. At autopsy,
uoxetin and noruoxetine were several fold higher than expected
and drug intoxication was stated as cause of death. Genetic testing
revealed a gene defect at the CYP2D6 locus, which resulted in poor
metabolism of uoxetin and exonerated the suspected adoptive
parents [95].
The tricyclic antidepressant amitriptyline is metabolized by the
polymorphic CYP2D6 and CYP2C19 (Fig. 6). In a series of forensic
autopsies, Koski et al. [96] found positive correlations between the
proportion of hydroxylated metabolites and the number of
functional copies of CYP2D6 and between the proportion of
demethylated metabolites and the functional copies of CYP2C19.
Especially when a suicidal intent is being proven, CYP genotyping
was proposed as a useful tool to add valuable information to the
interpretation of forensic-toxicological results.
Hair analysis also provided inter-individual variations of
amitriptyline metabolism and correlated with CYP2C19 and
CYP2D6 polymorphisms [97].
Major pathways of doxepin metabolism involving polymorphic
CYP enzymes are described in Fig. 7. In a case of fatal doxepin
poisoning with an undetermined manner of death, a completely
non-functional CYP2D6 genotype (*3/*4) indicated a total absence
of CYP2D6 enzyme and suggested a PM phenotype [98]. The
doxepin concentration in blood was 2.4 mg/L (1680 times higher
than considered therapeutic in a living patient), the concentration
of nordoxepin 2.9 mg/L, and the doxepin/nordoxepin ratio 0.83,
the lowest found among 35 other post-mortem cases analysed
during the same year. In 20 doxepin poisonings, a ratio of at least
3.8 (up to 75) was found, with only one case having a ratio as lowas
2.0. No alcohol or other drugs were detected in the presented case.
The CYP2C19 genotype was determined to be that of an EM. The
high concentration of nordoxepin was not consistent with an acute
intoxication and it was assumed that the defective genotype
contributed to the death, possibly involving repeated high dosage
of doxepin. The manner of death was denoted accidental, not
suicidal.
A patient developed severe adverse effects and did not
experience any therapeutic effect of venlafaxine. Further investi-
gations indicated that the patient was a PM for CYP2D6, the most
important phase I enzyme to metabolize venlafaxine [99]. This
corroborated that polymorphisms in the CYP450 gene inuence
the metabolic activity of the corresponding enzymes, thus
affecting the subsequent serum drug levels and their metabolites.
In another study, fatal venlafaxine poisonings were demonstrated
F. Musshoff et al. / Forensic Science International 203 (2010) 5362 59
to be associated with a high prevalence of drug interactions, but
the relative CYP2D6 activity did not predispose to high venlafaxine
concentrations [100]. Kobylecki et al. [101] also recently found no
signicant association between substance abuse disorder and
suicidal behaviour with the CYP2D6 and CYP2C19 genotypes
among patients with schizophrenia genotypes.
Of all the new antiepileptic drugs, only phenytoin undergoes
signicant metabolism by cytochrome P450 isozymes with signi-
cant genetic polymorphisms (CYP2C9, CYP2C19). According to
Anderson [102], studies are still needed to identify genetic and
biomarkers to identify patients at risk for serious idiosyncratic
reactions. Identication of experimental and clinical evidence
linking functional changes associated with gene mutations to
epilepsy syndromes would help to provide new molecular targets
for future antiepileptic drugs.
Clinically relevant pharmacokinetic drug interactions with
antidepressants are the object of recent research [103105] as are
pharmacogenetics in analgesia [2,106108] as well as in other areas
of medication. Relevant ndings should be taken into consideration
in forensic toxicology, where many of these substances are of
interest in daily routines.
4.5. Carisoprodol
The centrally-acting skeletal muscle relaxant carisoprodol is
metabolized to meprobamate by CYP2C19. In cases of driving under
the inuence of drugs the carisoprodol-to-meprobamate ratio
reects the number of active CYP2C19 alleles, indicating a gene-
dosage effect [109].
5. Conclusion
Pharmacogenetic analysis in forensic settings and especially in
post-mortem toxicology may reveal new aspects in daily routines.

Fig. 6. Major metabolic pathway of amitriptyline.

Fig. 7. Major pathways of doxepin metabolisminvolving polymorphic CYP enzymes.

F. Musshoff et al. / Forensic Science International 203 (2010) 5362 60
Therefore, an approach of molecular analysis including DNA
genotyping, together with forensic-toxicological analyses, can be
viewed as progress. In post-mortem cases in particular, these
approaches, together with macroscopic and microscopic scrutiny,
can be of relevance in modern medicolegal investigations. Because
other so-called invisible diseases like heart and liver abnormali-
ties can also be ascribed to genetic defects, post-mortem genetic
testing and the termmolecular autopsy could nd their way into
forensic practice [7,110,111].
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