药物和化学物毒性反应的遗传基础
AbstractInter-individual drug effects are subject to substantial
variability. There are multiple reasons based on
pathophysiological factors and environmental
interactions, but also genetic characteristics.
Groundbreaking successes have been achieved in the field
of pharmacogenomics and toxicogenomics. In particular,
the identification of hereditary polymorphisms in genes
of the cytochrome P450 system and phase II-enzymes such
as TMPT contributed considerably to the explanation of
the individually varying pharmacokinetics of a number of
drugs. Furthermore, hereditary variations in genes of
membrane drug transporters were recently discovered.
Along with these factors, which could influence
pharmacokinetics, strong efforts have been undertaken to
clarify the role of genetic polymorphisms in receptors
or signal transduction proteins modulating drug
efficacy. Particularly for malignant diseases such as
bladder or lung cancer, polymorphic foreign compound
metabolizing enzymes have been identified as
susceptibility factors, modulating an individual's
cancer risk dependent on the extent of environmental
exposure.
This review focuses on the role of the polymorphic phase
I enzymes cytochrome P450 1A1, 1A2, 1B1, 2C9, 2C19, 2D6,
3A5 and myeloperoxidase as well as on the phase
II-enzymes arylamine N-acetyltransferases 1 and 2,
glutathione S-transferases M1 and T1, and thiopurine
S-methyltranferases as detoxifying but also toxifying
factors, modulating pharmacokinetics and disease
susceptibility.
Keywords: Drug metabolism; Idiosyncratic reactions;
Toxification; Pharmacogenetics; Cytochrome P450
1. Introduction
The inter-individual effects of drugs and xenobiotics
are based on pathophysiological factors and
environmental interactions, but also genetic
characteristics. In many cases, toxicity depends on the
concentration at the side of action but may also be
subject of idiosyncrathic reactions. Despite iatrogenic
or patients failures, over-dosage may be the result of
impaired elimination due to organ failures, drug
interactions or hereditary consequences. On the other
side, the same metabolizing enzymes may contribute to
toxification of environmental compounds or
bio-activation of pro-drugs. Thus induction of such
pathways, e.g. Ah-receptor mediated enhancement of
cytochrome P4501A1 activity may lead to elevated
formation of reactive intermediates. These pathways,
however, have been also shown to be subject of genetic
variability. Aside the polymorphic drug metabolism,
there is increasing evidence that genetic differences in
membrane transporters may contribute to the explanation
of inter-individual variability of susceptibility of
adverse drug reactions or susceptibility to
xenobiotic-related diseases.
strong efforts have been undertaken to clarify the role
of genetic polymorphisms in receptors or signal
transduction proteins modulating drug efficacy, as well
as in factors involved in cancer etiology such as
factors controlling cell cycle, apoptosis and DNA
repair.
The broad field of research within pharmacogenomics is
trying to elucidate the complex interaction of these
polymorphic genes in order to explain and to develop
improved therapies particularly for common illnesses
such as cardiovascular und malignant diseases and to
reduce the number of adverse drug events (Evans and
Relling, 2004). This is both reasonable and necessary
for several rationales; e.g. a prospective study in the
USA points to the fact that 6.7% of hospitalisations can
be traced to adverse drug side effects (Lazarou et al.,
1998). The resulting costs in the USA have been
estimated to amount yearly between 30 and 100 million
US$ (White et al., 1999). Pharmacogenomics offers the
opportunity, on one hand, to identify new drug targets,
and, on the other hand, to adjust individually the
required dosage of drugs available on the market
(Kirchheiner et al., 2005).
This review focuses on the role of polymorphic drug
metabolizing enzymes in the toxicity of drugs and other
chemicals.
2. Adverse effects and toxicity due to impaired
detoxification pathways
2.1. Cytochrome P450s
The phase I-enzymes catalyze oxidative and reductive
reactions of the foreign compound metabolism, but also
of transformation of certain lipids and steroids.
Obviously, cytochrome P450s are most important and the
CYP3A-family contributes to approximately 50% of the
total cytochrome P450 activity of the adult human liver.
It metabolizes about 60% of all usually prescribed
drugs. However, nearly 30% of all drugs are
polymorphically metabolized in the human liver
particularly by cytochromes P450 (CYP) 2C9, 2C19, 2D6
(Table 1). There are many other polymorphisms known in
P450s enzymes such as 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2E1,
3A4, 3A5, 3A7, 5A1, and 8A1, but the functional
significance towards drug metabolism is discussed
controversial (Ingelman-Sundberg, 2001a).
Table 1. Substrates of polymorphic cytochrome P450
enzymes, functional important alleles and frequencies in
Caucasiansa
CYPDrugs (selected)ChemicalsAllele (relevant DNA
and/or protein variant)Functional
consequenceAllele frequency (%)b
CYP1A1 Polycyclic hydrocarbons*2A (3801T > C)Not
confirmed4–7
*2B (2455A > G, I462V; 3801T > C)Not confimed3
*4 (2453C > A, T461N)
Not confirmed3
CYP1A2Various: theophylline, caffeine,
fluvoxamine, mexiletineArylamines*1C (−3860G >
A)Decreased activity33
*1F (−163C > A)Induced activity66
*1K (−729C > T)Decreased activity1–3
CYP1B1 Polycyclic hydrocarbons*2 (142C > G, R48G;
355G > T, A119S)Not confirmed30
Not confirmed
*3 (4326C > G, L432V)Not confirmed44
*4 (4390A > G, N453S) 18
CYP2A6Various: cumarines, nicotine *2 (T479A,
L160H)Inactive enzyme1–3
*4 (Gene deletion)No enzyme1
*9 (−48T > G)Decreased expression5
CYP2B6Cytostatics: cyclophosphamide, ifosfamide,
benzodiazepines: diazepam, tenazepam, midazolam.
Various: clopidogrel, nicotine, tamoxifen *5
(1459C > T, R487C)Decreased activity14
*7 (516G > T, Q172H; 785A > G, K262R; 1459C >
T, R487C)Decreased activity1
CYP2C8Various: paclitaxel, rosiglitazone *2 (805A
> T, I269F)Decreased activity0
*3 (416G > A; R139K; 1196A > G, K399R)Decreased
activity13
CYP2C9NSAIDs: diclofenac, ibuprofen, S-naproxen,
meloxicam, piroxicam, tenoxicam. Oral
antidiabetics: glibenclamide, glipizide,
tolbutamide AT1-antagonists: irbesartane,
losartane, valsartane. Various: cyclophosphamide,
amitriptyline, fluoxetine, phenytoine,
sulfamethoxazole, tamoxifen, torasemide,
S-warfarin *2 (430C>T, R144C)Decreased
activity8–13
*3 (1075A > C, I359L)Decreased activity7–9
CYP2C19Proton pump inhibitors: lansoprazole,
omeprazole, pantoprazole, anticonvulsants:
diazepam, phenytoine, S-mephenytoine
antipsychotics: citalopram, clomipramine,
imipramine. Various: cyclophosphamide,moclobemide,
proguanil, propranolole *2 (681G > A, splice-site
mutation)Inactive enzyme13
*3 (636G > A, Stop)
Inactive enzyme0
CYP2D6β-Blockers: carvedilol, metoprolol,
propranolol. Antiarrhythmics: propafenone,
encainide, flecainide, mexiletine, sparteine.
Neuroleptics: haloperidol, perhexiline,
perphenazine, risperidon, thioridazine.
Antidepressants: amitriptyline, clomipramine,
desipramine, fluoxetine, fluvoxamine, imipramine,
maprotiline, nortriptyline, paroxetine. Various:
codeine, dextromethorphan, tramadol amphetamine,
debrisoquine, ondansetron,
phenacetineNitrosamines*2xN (gene
duplication)Increased activity1–5
*2 (R296C; S486T)Slightly decreased act.12–21
*4 (1846G > A splice-site mutation)Inactive
enzyme
*5 (gene deletion)
*6 (1707T > del frameshift)No enzyme4–6
*10 (P34S)No enzyme1
*17 (T107I; R296C)Decreased activity1–2
*41 (−1584G > C)Decreased activity<1
Decreased expression10–20
CYP2E1Anaesthetics: enflurane, halothane,
isoflurane, methoxyflurane, sevoflurane. Various:
paracetamol, chlorzoxazone, ethanolHalogenated
aliphates*2 (R76H)Decreased activity0
*3 (V381I)Inactive enzyme<1
*4 (V179I)Inactive enzyme<1
CYP3A5Widely overlapping with CYP3A4 substrates *3
(intron 3 6986A > G splice-site mutation)No
enzyme94
a Data according to (Cascorbi et al., 1996,
Ingelman-Sundberg, 2001b, Lang et al., 2001,
Rylander-Rudqvist et al., 2003 and Sachse et al., 1997).
b Among Caucasians
2.1.1. Cytochrome P4592C9
There is increasing evidence that the bleeding risk of
patients, treated with the vitamin K-antagonist warfarin
for thrombosis prophylaxis is increased among carriers
of a low active variant of cytochrome P4502C9 (CYP2C9)
(Aithal et al., 1999). Particularly the effective
S-enantiomer of warfarin is significantly decreased in
these individuals, resulting in augmented intermediate
plasma concentrations. Consequently, the synthesis of
the vitamin K-dependent coagulation factor is strongly
inhibited, corresponding to elevated INR values and
increased tendency to bleeding episodes (Daly and King,
2003).
Cytochrome P450 2C9 belongs to a close gene cluster on
chromosome 10, comprising CYP2C8, 2C9, 2C18, and 2C19.
The CYP2C9 deficiency is determined to a major extent by
two missense point mutations that lead to the exchange
of the amino acids Arg144Cys (CYP2C9*2) and Ile359Leu
(CYP2C9*3) (Goldstein and de Morais, 1994). The allele
frequencies in the Caucasian population are
approximately 11% or 7%, respectively. Homozygous
carriers who account for the phenotype of poor
metabolizes have a prevalence of about 3–4%.
Interestingly in Asians, an alternative Ile359Thr amino
acid replacement was observed, termed CYP2C9*4 (Imai et
al., 2000). Further polymorphisms were identified in
African-Americans (Dickmann et al., 2001 and Kidd et
al., 2001). However, the phenomenon of vitamin
K-antagonists resistance is also due to genetic
polymorphisms. Recently it was shown that subjects,
providing variants in the gene of the vitamin K epoxide
reductase complex, subunit 1 (VKORC1) require
significantly higher warfarin doses than usually
recommended (D’Andrea et al., 2005 and Yuan et al.,
2005).
Beside warfarin (but to a much lower extent
phenprocoumon), many non-steroidal antiphlogistics such
as diclofenac, ibuprofen, and meloxicam, oral
antidiabetics like tolbutamide and glimenclamide, the
angiotensin receptor antagonists irbesartan and losartan
as well as some other medications such as phenytoin are
metabolized by CYP2C9 (Kirchheiner and Brockmoller,
2005). Indeed, cutaneous adverse reactions during
phenytoin therapy account in part to the CYP2C9 genotype
(Lee et al., 2004). Moreover, there is increasing
evidence that the clearance of oral antidiabetics is
directly dependent on the CYP2C9 genotype. The
consequences are not always clear. After glubyride or
glibenclamide intake, the area under the curve increased
nearly three-fold, but glucose levels of heterozygous
carriers of CYP2C9 variants did not differ (Niemi et
al., 2002). In homozygous CYP2C9*3-carriers, these
effects are more pronounced and are mirrored in
accelerated insulin response to glucose stimulation
(Kidd et al., 1999 and Kirchheiner et al., 2002).
2.1.2. Cytochrom P450 2C19
CYP2C19 (mephenytoine hydroxylase) catalyzes the
hydroxylation particularly of proton pump inhibitors
like omeprazole and lanzoprazole, but not rabeprazole.
Beside many other variants, a splice-site mutation leads
to total lack of any activity in 3–5% of Caucasians (De
Morais et al., 1994). Interestingly, there is increasing
evidence that poor metabolizers profit much stronger
from helicobacter pylori eradication and
gastroesophageal reflux therapy than extensive
metabolizers (Tanigawara et al., 1999, Kawamura et al.,
2003, Schwab et al., 2004 and Furuta et al., 2005). On
the other hand, the elimination of the alkylating
cytostatic cyclophosphamide is significantly impaired in
CYP2C19 poor metabolizers (Timm et al., 2005). The
clinical consequences remain currently open. Moreover,
certain antidepressants are metabolized at least in part
by CYP2C19. Many novel SNPs have been detected recently
and currently there are 19 different alleles annotated,
but most of the genetic variants identified have a very
low prevalence. Aside the G681A splice site mutation,
G636A (CYP2C19*3) should be considered genotyping Blacks
and Orientals. However, this premature stop codon is
rare in Caucasians.
2.1.3. Cytochrome P450 2D6
CYP2D6 is one of the best characterized cytochrome P450
enzymes (for review see Bertilsson et al., 2002).
Approximately 7–10% of the European populations are
CYP2D6-poor metabolizers who show reduced metabolism of
numerous drugs like antiarrhythmics, antidepressents,
neuroleptics, and some betablockers or opiates. Among
this subgroup, clinically relevant drug side effects are
more likely compared to extensive metabolizers. The
occasionally observed absence of the desired effect
accounts in some cases to the phenomenon of gene
duplications, occurring with 1–3% in Middle-Europeans.
CYP2D6 belongs to a gene cluster on chromosome 22q13.1
of the highly homologues inactive pseudogenes CYP2D7
containing a single reading frame-disrupting insertion
in its first exon and the real pseudogene CYP2D8
(Eichelbaum et al., 1987, Gough et al., 1993 and Kimura
et al., 1989). The polymorphisms are well characterized
and extensively described by Sachse et al. (1997).
Currently more than 90 different CYP2D6 haplotypes are
recorded by the human cytochrome P450 (CYP) allele
nomenclature committee
([url]http://www.imm.ki.se/CYPalleles[/url]). The alleles may be
classified into functional, non-functional and reduced
function groups. One of the major primary gene defect at
the cytochrome P450 CYP2D locus is a 1846G > A splice
site mutation (CYP2D6*4) (Gough et al., 1990) with a
frequency of 20.7% in Caucasians (Sachse et al., 1997).
In 4–6% of Caucasian and other ethnic populations, the
entire coding region is deleted (Gaedigk et al., 1991).
Hence, allele *5 is believed to have an ancient origin.
Further relevant variants are a 2549A deletion in *3
(2.0%), and a 1707T deletion in *6 (0.9%), generating
frame shifts. In contrast to these fatal polymorphisms,
a triple-base-pair deletions in allele *9 (1.8%) does
not significantly alter enzyme activity (Broly and
Meyer, 1993) and a proline to serine exchange in codon
34 is associated with lower enzyme activity and
particularly decreased stability of CYP2D6.10 (Nakamura
et al., 2002). This variant occurs with 1–2% in
Caucasians, but is the major cause of low CYP2D6
activity in Orientals (Bertilsson, 1995). In
African-Blacks, *17 is one of the major reasons for low
CYP2D6 activity.
The intermediate phenotype is also due to diminished
expression rates of CYP2D6, e.g. there is convincing
evidence that homozygous carriers of a C/G polymorphism
1584 bp upstream of the start codon (CYP2D6*41)
exhibited only 50% of protein compared to carriers of
the −1584G variant (Zanger et al., 2001).
Extremely high CYP2D6 activities in 1–2% of Caucasians
were identified to be due to gene duplications of the
wild-type and allele CYP2D6*2 but possibly also of
others. In Northern Europe the prevalence is below 2%
(Dahl et al., 1995), but in some regions of Spain,
frequencies of more than 7% were observed (Agundez et
al., 1995). In Arabian countries (McLellan et al., 1997)
as well is in the North-East-African Ethiopia, a
prevalence of ultra rapid metabolizers of up to 29% is
reported (Aklillu et al., 1996). In a few cases, there
were families with up to thirteen gene copies
identified. In contrast, in China, CYP2D6 gene
duplications are absolutely rare, but the mean metabolic
ratio of debrisoquine/4-hydroxydebrisoquine is increased
compared to Caucasians (Johansson et al., 1994). This is
due to the high prevalence of the low active
(intermediate) CYP2D6*10 variant (Garcia-Barcelo et al.,
2000). In Blacks, however, large heterogeneity seems to
exist (Griese et al., 1999 and Masimirembwa et al.,
1996).
The ultrarapid metabolizer phenotype of CYP2D6 has been
well established as a relevant cause of non-response to
antidepressant drug therapy. Clearance of such drugs
like nortriptyline, desipramine, and to some extent
imipramine and amitriptyline (Brosen et al., 1991,
Ghahramani et al., 1997 and Venkatakrishnan et al.,
1999) evidently depend on the CYP2D6 polymorphism.
Specific serotonine reuptake inhibitors like fluoxetine,
citalopram or paroxetine were shown to be inhibitors of
CYP2D6 (Alfaro et al., 1999 and Crewe et al., 1992). The
effects of the CYP2D6 polymorphism on antipsychotic
therapy appear to be more pronounced in neuroleptics.
Compounds like perphenazine, zuclopenthixol,
thioridazine, haloperidol and risperidone are
metabolized to a significant extent by CYP2D6. Poor
metabolizers appeared to posses an elevated risk to
suffer from side effects like extrapyramidal symptoms
(Bertilsson et al., 2002, Brockmoller et al., 2002 and
Scordo et al., 2000). Moreover, the antipsychotic
efficacy seems to be influenced by the number of active
copies of CYP2D6 genes (Brockmoller et al., 2002). The
findings give rise to perform genotyping before
treatment with polymorphically metabolized
antipsychotics (Dahl, 2002).
Betablockers like metoprolol, and to some extent
carvedilol are also CYP2D6 substrates (Oldham and
Clarke, 1997, Pepper et al., 1991 and Rau et al., 2002).
However, currently there is no data available if the
clinical outcome of betablocker therapy is influenced by
genetic polymorphisms of CYP2D6.
The beneficial use of CYP2D6 genotyping for drug therapy
was demonstrated recently on the treatment of nausea and
vomiting in cancer chemotherapy with the antiemetic
5-HT3 receptor antagonist tropisetron (Kaiser et al.,
2002). In this case, CYP2D6 poor metabolizer took
advantage of antiemetic therapy compared to extensive
metabolizers.
2.1.4. Cytochrome P450 3A
The CYP3A-family consists of the well known, numerous
drugs metabolizing CYP3A4, of CYP3A5, of the fetal
CYP3A7, as well as of the recently identified CYP3A43.
The interindividual variability of the total
CYP3A-activity accounts in part to the presence or
absence of active CYP3A5. Only less than a third of
Caucasians and 66% of African-Blacks exhibit CYP3A5
expression, caused by a G/A-DNA base exchange within
intron 3. This single nucleotide polymorphism leads to
alternative splicing and generation of a premature stop
codon in exon 3B, resulting in the inactive CYP3A5*3
(Hustert et al., 2001a and Kuehl et al., 2001). This
polymorphism explains in part the bidomal distribution
of midazolam kinetics. Additionally among
American-Blacks, a rare G/A-SNP was identified in exon
7, associated with reduced CYP3A5 activity (CYP3A5*6)
(Kuehl et al., 2001).
Aside CYP3A5, CYP3A4 contributes to the clearance of
midazolam. Recently, a functional significant
polymorphism of CYP3A4 (L373F) was discovered. It
reduces affinity of midazolam to CYP3A4 from a KM of 8.7
μmol/l to a KM of 36.4 μM. Further novel identified SNPs
lead in part to lowered or even lack of expression.
However, due to the rarity of these polymorphisms, they
do not contribute to the explanation of individual
variability of CYP3A4 activity (Eiselt et al., 2001).
CYP3A can be strongly induced by drugs like rifampicin,
carbamazepine, phenobarbital and others. They interact
with the nuclear pregnane X receptor (PXR) followed by
dimerization with the 9-cis-retinoic acid receptor α
(RXRα) and binding to the respective promoter responsive
elements. Therefore genetic variants of the PXR gene
could contribute to the variability of the CYP3A
activity. Indeed, PXR exhibits numerous variants, at
least six of them generate amino acid exchanges. The
variant 163G leads to a 15-fold induction by rifampicin
compared with a five-fold induction by 163D. Other
variants such as V140M and A370T exhibit effects of
minor significance (Hustert et al., 2001b). These
discoveries show the great importance of
gene–environmental interactions, since the PXR
polymorphisms do not affect the basal CYP3A activity,
but the CYP3A induction. Likewise CYP3A4 polymorphisms,
the prevalence of PXR SNPs is rather low, hence the
observed CYP3A activity can be described only to a small
part by this variables.
2.2. Phase II-enzymes
Major enzymes of conjugation in Phase II are the UDP
glucuronosyltransferase (UGT), sulfotransferases (SULT),
arylamine N-acetyltransferases (NAT), glutathione
S-transferases (GST), thiopurine S-methyltransferases
(TPMT), catecholamine-O- methyltransferases (COMT) and
others. The polymorphic character of NAT and GST and its
role towards particularly towards malignancies is
extensively investigated and the role of TPMT for
azathioprine toxicity is well established. The role of
polymorphisms of sulfotransferases is much more
difficulty to consider, since the different isoforms of
sulfotransferases are involved in detoxification, but
also toxification pathways, leading to partly
contradictory results (excellently reviewed by Glatt and
Meinl (2004)). Also COMT is polymorphic and was related
to neuro-psychiatric disorders (Glatt et al., 2003 and
Redden et al., 2005) or malignancies (Mitrunen and
Hirvonen, 2003), the associations are, however, weak or
need confirmation. Also the UGT family provides certain
polymorphic traits, hereditary defects may lead to mild
or severe hyper-bilirubinemia, and there is increasing
evidence that genetic variants may have an important
pharmacological impact on e.g. anti-cancer therapy with
irinotecan (Ando et al., 2000). For review see
(Burchell, 2003).
2.3. Arylamine N-acetyltransferases
Arylamine N-acetyltransferases are responsible for the
conjugation of drugs like isoniazid, dapsone,
procainamide and many others. The slow NAT2 acetylator
is supposed to be at higher risk for drug side effects
such as peripheral neuropathia after isoniazid treatment
(Yamamoto et al., 1996) or certain disorders such as
drug-induced lupus erythematosus and Stevens–Johnsons
syndrome (Wolkenstein et al., 1995). This severe
diseases may be caused, in susceptible individuals, by a
large number of drugs involved in acetylation
metabolism, the there is no association to the
idiopathic form of LE (Zschieschang et al., 2002).
Peripheral neuropathy provoked by isoniazid over-dosage
may be a major problem in ethnicities with a high
frequency of slow acetylators as in Northern Africa. Low
drug efficacy may be expected in rapid acetylators. NAT2
is expressed preferably in the human liver, whereas the
sister gene NAT1 can be determined in a wide variety of
different tissues.
Phenotyping studies in the last four decades disclosed
distinct ethnic differences of slow acetylator
frequencies (Evans, 1992). Extremes can be found between
North Africa with a frequency of alleles coding for slow
acetylation of 95% and the Far East Pacific region with
a frequency of only 11%. The expected partition of slow
acetylators in these populations spans 90–1.2% (Cascorbi
et al., 1995, Ilett et al., 1993 and Lin et al., 1993).
In African Blacks, there is an additional frequent 191G
> A SNP, and a large diversity of haplotypes can be
observed (Cascorbi et al., 1999).
The implication of both, oxidative metabolization by
cytochrome P450s and conjugation with acetyl-CoA also
plays a major role in an individual's risk to suffer
from certain diseases, which are related to
environmental toxicants, particular cancer.
Earlier phenotyping studies provided some evidence that
slow acetylators are at increased risk for bladder
cancer (Cartwright et al., 1982, Lower et al., 1979 and
Vineis and Ronco, 1992). Later studies performed using
genotyping methodologies confirmed these early findings
(Brockm鰈ler et al., 1996 and Risch et al., 1995). It is
hypothesized that in rapid acetylators arylamines, as
contained in aniline dyes or cigarette smoke (e.g.
4-aminobiphenyl), are detoxified by N-acetylation in the
liver and excreted in the urine. In contrast, low
N-acetylation activity leads to increased formation of
N-hydroxylated products. These hydroxylamines may
undergo further O-acetylation in the urinary bladder
preferentially by arylamine N-acetyltransferase 1
(NAT1), which was found to be expressed in the urinary
epithelium (Kloth et al., 1994). The product, arylamine
acetoxyesters are unstable in the acid environment and
disintegrate spontaneously to arynitrenium ions. These
highly reactive radicals may well interact with proteins
and DNA of bladder epithelial cells forming adducts
(Hein et al., 1993).
A number of in vitro studies gave evidence for this
theory. Most carcinogenic compounds activated by
acetyltransferases are heterocyclic aromatic amines like
2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP)
which lead to dose-dependent effects in mutagenicity
tests (Wild et al., 1995).
A meta-analysis of 22 published case-control phenotyping
and genotyping studies conducted in a total of 2496
cases and 3340 controls in different populations
revealed that slow acetylators had a 40% increased risk
compared to rapid acetylators (odds ratio 1.4, 95%
confidence interval 1.2–1.6) (Marcus et al., 2000). In
particular, the largest genotyping studies clearly
showed a gene-environment interaction (Vineis et al.,
2001).
Aside from the unequivocal role of NAT2, the discovery
of the polymorphic nature of NAT1 raised the question
whether different NAT1 genotypes may additionally
modulate bladder cancer susceptibility. Indeed, (Bell et
al., 1995) reported two important facts: NAT1*10 was
found to provide enhanced activity in bladder tissue
compared to NAT1*4 and moreover, the frequency of
NAT1*10 was increased among bladder cancer patients.
However, these results are conflicting (Okkels et al.,
1997) and recently we were able to show that NAT1*10
does not alter enzyme activity towards ex vivo formation
of N-acetyl p-amino benzoic acid (Bruhn et al., 1999).
We found a significant decrement of NAT1*10 genotypes
among 425 bladder cancer patients; the adjusted odds
ratio was 0.65 (95%-C.I. 0.46–0.91; P = 0.013) (Cascorbi
et al., 2001). Considering the NAT2 genotype, a clear
under-representation of NAT1*10 genotypes among rapid
NAT2 genotypes in the cases studied (odds ratio 0.39;
95%-C.I. 0.22–0.68; P = 0.001), and a
gene–gene–environment interaction was observed.
NAT2*slow/NAT1*4 genotype combinations with a history of
occupational exposure were six times more frequent in
cancer cases than in controls without risk occupation (P
< 0.0001).
Similar as in the bladder, one of the first steps of
colon cancer may be initiated by DNA adducts, formed by
heterocyclic arylamines, which had been activated by
cytochromes P450s and N-acetyltransferases. It is well
established that NAT1 as well as NAT2 are expressed in
colon tissue (Hickman et al., 1998). Enhanced
O-acetylation in the colon mucosa could therefore
contribute to the formation of adducts (Nerurkar et al.,
1995). Indeed, some studies suggested the rapid
acetylators phenotype as a hereditary trait for
predisposition of colorectal carcinomas particularly
when patients had a history of smoking or red meat
intake. (Chen et al., 1998, Roberts-Thomson et al., 1996
and Welfare et al., 1997). Independently of confounders,
extended risk was observed rapid acetylators (Agundez et
al., 2000 and Gil and Lechner, 1998). However, the
findings on NAT2 as a susceptibility factor of colon
cancer are not consistent less pronounced in several
other large molecular epidemiological studies (Slattery
et al., 1998).
2.3.1. Thiopurine-S-methyltransferase (TPMT)
A rare but serious side effect in treatment with the
antimetabolites 6-mercatopurine and azathioprine is a
severe bone marrow depression, which may result in
lethal side effects (Krynetski and Evans, 2000).
Detoxification takes place by TPMT, a phase-II enzyme,
which however, is homozygously deficient in one of 300
individuals (Weinshilboum, 1992). Among these patients,
metabolism takes place by an alternative pathway to the
6-thioguanin-nucleotide (6-TGN). The plasma
concentration of 6-TGN correlates with the severity of
the medication's side effects. However, a problem
arises, since TPMT exhibits a high number of mutations,
which allows a genotyping only to a limited degree. So
far, eight mutations that determine an amino acid
exchange and a splice site mutation are known.
Apparently even more, though very rare, SNPs exist which
determine a TPMT-poor metabolizer. Many clinics still
routinely prefer ex-vivo phenotyping procedures to date,
but increasing knowledge about rare variations and
application of techniques like DHPLC will allow a
reliable prediction of TMPT status by means of
genotyping (Schaeffeler et al., 2001). The clinical
importance was shown in a large study in pediatric acute
lymphoblastic leukemia patients, showing that TPMT-poor
metabolizers are at clear risk of severe azathioprine
side effects, making a significant dose reduction
necessary (Stanulla et al., 2005).
2.3.2. Glutathione S-transferases
Glutathione S-transferases of classes GSTA, -M, -P, -T,
and -Z are conjugating a variety of exogenous and
endogenous compounds including several cytostatics.
Since glutathione-conjugation represents a
detoxification pathway, it becomes rapidly clear that
the total absence of GSTM1 activity (genotype
GSTM1*0/*0) may be linked to increased drug toxicity or
cancer susceptibility. Indeed, GSTM1 deficiency was
shown by several studies to be a risk factor for lung
(Houlston, 1999), laryngeal (Hashibe et al., 2003) and
urinary bladder cancer (Brockmoller et al., 1994 and
Engel et al., 2002), comprehensively reviewed by Parl
(2005). Howewer, there is a clear lack of association
between GSTM1, GSTT1, and GSTP1 and breast cancer (Vogl
et al., 2004).
These findings may be partly explained due to the fact
that glutathione S-transferase deficiency prevents from
detoxification of dioepoxide from e.g. benzo(a)pyrene.
Thus several studies could show elevated levels of DNA
and protein adducts in carriers of GSTM1 and GSTT1
(Alexandrov et al., 2002 and Bartsch et al., 1999).
Interestingly GSTM1 deficiency was also reportedly
associated with other smoking-related diseases such as
atherosclerosis (Olshan et al., 2003) or lung emphysema.
3. Toxicity due to elevated toxification
This chapter deals with the role of polymorphic
metabolizing enzymes in the activation of parent
compounds to highly active intermediates, hence less
active phenotypes would lower the risk of e.g. DNA aduct
formation, whereas polymorphisms leading to elevated
activity would be potentially associated with an
increased risk of e.g. tobacco-smoke related cancers.
3.1. Cytochrome P450s
Bioactivation of xenobiotics accounts to some extend to
the inducible cytochrome P450s 1A1, 1A2, and 1B1. CYP1A1
is a key enzyme in carcinogen metabolism and was proved
as a promising genetic biomarker for susceptibility to
certain malignancies, particularly lung cancer (Vineis
et al., 2004). It metabolizes polycyclic aromatic
hydrocarbons such as benzo[a]pyrene (BaP), a prominent
and highly carcinogenic polycyclic aromatic hydrocarbon
(PAH) present in tobacco smoke, into benzopyrene diol
epoxide (BPED) which reacts with DNA predominantly at
the N2-position of guanine to produce primarily
N2-guanine lesions, e.g., BPDE-N2−dG adduct (Osborne,
1990). CYP1A1 is highly polymorphic, but most data is
available on a T to C-transition 1194 bp downstream of
exon 7, creating a new MspI-cleavage site at position
3801T > C (CYP1A1*2A). A meta-analysis by Vineis et al.
(2003) revealed that smokers, homozygous for 3801C were
at significantly elevated risk of lung cancer (odds
ratio 2.36). However, an Ile462Val exchange is in strong
linkage disequilibrium and the haplotype CYP1A1*2B was
shown to be associated to lung cancer even in
non-smokers (Raimondi et al., 2005). The particular role
of these polymorphisms considering also the GSTM1
genotype, was shown for significantly elevated DNA
adduct levels in lymphocytes and lung cancer tissue
(Rojas et al., 2000 and Rojas et al., 2004). A further
adjacent Thr461Asp amino acid replacement revealed no
evidence of an association to lung cancer (Cascorbi et
al., 1996), but this SNP modulates the substrate
affinity particularly of 17beta-estradiol and estrone,
giving rise to a possible involvement in hormone-related
cancers (Kisselev et al., 2005 and Schwarz et al.,
2001).
The role of the polymorphic CYP1B1 seems to be less
important as cancer susceptibility factor. Although
CYP1B1 is involved in estrogen metabolism, there is lack
of evidence for an association to breast cancer in a
recent large meta-analysis (Wen et al., 2005).
3.2. Myeloperoxidase
Myeloperoxidase (MPO) has been shown to transform
environmental precarcinogens such as benzo(a)pyrene and
aromatic amines to highly reactive intermediates
(Kadlubar et al., 1992 and Mallet et al., 1991). MPO is
a lysosomal hemoprotein expressed in polymorphonuclear
leukocytes and monocytes that actually catalyzes the
production of hypochlorous acid in physiologic
situations which leads to microbicidal activity against
a wide range of organisms (Foster et al., 1998).
Strkingly, we found an association of MPO genotypes and
clozapin-induced agranulocatosis, a severe and
treatment-limiting side effect of treatment with this
atypical neuroleptic drug (Mosyagin et al., 2004). On
the other hand, the highly active −463G allele is
strongly associated to lung cancer (Cascorbi et al.,
2000 and Schabath et al., 2002), indicating elevated
formation of reactive intermediates, leading to DNA
adducts, as could be demonstrated in bronchoalveolar
lavage fluid; cytology (Van Schooten et al., 2004).
4. Conclusion
The early adaptation of a therapy regimen to genetic
traits could help to avoid side effects and improve the
clinical outcome of pharmacotherapy. Genotyping instead
of phenotyping with probe drugs should be preferred
during an on-going pharmacotherapy, since some drug may
inhibit e.g. CYP activity. For prediction of the
phenotype by genotyping at least the major deficient
alleles should be characterized, occurring in the
particular ethnic group. The benefit of genotype-realted
dose-adaption is best described by studies of
psychotropic drugs (Bertilsson et al., 2002), however
there is a need of proof performing prospective studies
(Brockmoller et al., 2000). First dosage recommendations
in dependence of pharmacogenetic traits have been
published recently for a set of antidepressants
(Kirchheiner et al., 2001). This may be an important
step in the attempt to improve individual drug therapy,
but more standardized clinical studies are required,
testing the efficacy and side effects of
genotype-adapted and non-adapted dosage regimens.
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