22
Do Cytochrome P450 Enzymes Contribute to the
Metabolism of Xenobiotics in Human?
1Khaled
1Pharmacology
Abass1*, Petri Reponen1,2, Miia Turpeinen1,
Sampo Mattila2 and Olavi Pelkonen1
and Toxicology Unit, Institute of Biomedicine, University of Oulu
2Department of Chemistry, University of Oulu
Finland
1. Introduction
The cytochromes P450 (CYP) comprise a large multigene family of hemethiolate proteins
which are of considerable importance in the metabolism of xenobiotics and endobiotics. CYP
enzymes in humans as well as in other species have been intensively studied during recent
years (Pelkonen et al., 2008; Turpeinen et al., 2007). It is possible to characterize metabolic
reactions and routes, metabolic interactions, and to assign which CYP is involved in the
metabolism of a certain xenobiotic by different in vitro approaches (Pelkonen et al., 2005;
Pelkonen & Raunio, 2005; Hodgson and Rose, 2007a). Risk assessment needs reliable scientific
information and one source of information is the characterization of the metabolic fate and
toxicokinetics of compounds. Toxicokinetics refers to the movement of a xenobiotic into,
through, and out of the body and is divided into several processes including absorption,
distribution, metabolism, and excretion (ADME). Metabolism is one of the most important
factors that can affect the overall toxic profile of a pesticide. During metabolism, the chemical
is first biotransformed by phase I enzymes, usually by the cytochrome P450 (CYP) enzyme
system, and then conjugated to a more soluble and excretable form by phase II conjugating
enzyme systems (Guengerich & Shimada, 1991). In general, these enzymatic reactions are
beneficial in that they help eliminate foreign compounds. Sometimes, however, these enzymes
transform an otherwise harmless substance into a reactive form – a phenomenon known as
metabolic activation (Guengerich & Shimada, 1991).Exposure to pesticides is a global
challenge to risk assessment (Alavanja et al., 2004; Maroni et al., 2006). On a world-wide basis,
acute pesticide poisoning is an important cause of morbidity and mortality. In an
extrapolation, WHO/UNEP estimated that more than 3 million people were hospitalized for
pesticide poisoning every year and that 220 000 died; it particularly noted that two-thirds of
hospitalizations and the majority of deaths were attributable to intentional self-poisoning
rather than to occupational or accidental poisoning (Konradsen et al., 2005; WHO/UNEP,
1990). Humans are inevitably exposed to pesticides in a variety of ways: at different dose
levels and for varying periods of time (Boobis et al., 2008; Ellenhorns et al., 1997).
1
* corresponding author email: khaled.megahed@oulu.fi; khaled.m.abass@gmail.com
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Fungicides
2. CYPs - the human xenobiotic-metabolizing enzymes
CYPs are found in high concentrations in the liver, but are present in a variety of other
tissues, including lungs (Lawton et al., 1990), kidneys ((Hjelle et al., 1986; Tremaine et al.,
1985), the gastrointestinal tract (Dutcher & Boyd, 1979; Peters and Kremers, 1989), nasal
mucosa (Adams et al., 1991; Eriksson and Brittebo, 1991), skin (Khan et al., 1989) and brain
tissue (Bergh & Strobel, 1992; Dhawan et al., 1990). CYPs are categorized into families and
subfamilies by their sequence similarities. Humans have 18 families of cytochrome P450
genes and 44 subfamilies. The enzymes are thus identified by a number denoting the family,
a letter denoting the subfamily and a number identifying the specific member of the
subfamily. The example given below explains the system of nomenclature followed (Fig. 1).
Fig. 1. An example of the nomenclature of the cytochrome P450 enzymes (modified from
(Wijnen et al., 2007)).
The website http://drnelson.utmem.edu/CytochromeP450.html contains more detailed
classification related to the cytochrome P450 metabolizing enzymes. The CYP enzymes in
families 1-3 (Fig. 2) are active in the metabolism of a wide variety of xenobiotics including
drugs (Pelkonen et al., 2008; Pelkonen et al., 2005; Rendic & Di Carlo, 1997).
Fig. 2. Relative abundance of individual CYP forms in the liver (modified from (Pelkonen et
al., 2008)).
2.1 CYP1A subfamily
CYP1A1 and CYP1A2 are members of the CYP1A subfamily. CYP1A1 is a major
extrahepatic CYP enzyme and its level of expression in human liver is very low (Pelkonen et
al., 2008; Guengerich & Shimada, 1991; Ding & Kaminsky, 2003; Edwards et al., 1998;
McKinnon et al., 1991; Pasanen & Pelkonen, 1994; Raunio et al., 1995). In humans, CYP1A2 is
expressed mainly in liver and in lower levels in lung along with CYP1A1 (Liu et al., 2003;
Wei et al., 2001; Wei et al., 2002). CYP1A2 represents about 10% of total CYP enzymes in the
human liver (Pelkonen & Breimer, 1994; Shimada et al., 1994). CYP1A2 enzyme levels in the
human liver display some variability between individuals (Shimada et al., 1994).
2.2 CYP2A subfamily
The human CYP2A subfamily contains three genes i.e. CYP2A6, CYP2A7, and CYP2A13,
and two pseudogenes (Hoffman et al., 1995; Honkakoski & Negishi, 1997; Pedro et al., 1995).
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
443
CYP2A6 represents 10% of the total CYP content in liver (Pelkonen et al., 2008; Yun et al.,
1991). CYP2A6 enzyme activity in the human liver displays a relatively large variability
between individuals, and some Japanese are known to lack the functional protein
completely (Pelkonen et al., 2008; Pelkonen et al., 2000; Shimada et al., 1996).
2.3 CYP2B subfamily
CYP2B6 represents approximately 1-10% of the total hepatic CYPs. A notable
interindividual variability in the expression of CYP2B6 has been reported (Code et al., 1997;
Faucette et al., 2000; Lang et al., 2001; Stresser & Kupfer, 1999; Yamano et al., 1989). CYP2B6
has a high polymorphic expression and it is affected by genotype and gender.
2.4 CYP2C subfamily
The CYP2C subfamily has four active members, namely 2C8, 2C9, 2C18 and 2C19. CYP2Cs
are the second most abundant CYP proteins in human liver and the CYP2C subfamily consists
of three members, comprising about 20 % of the total P450 enzymes. In humans, CYP2C9 is the
main CYP2C, followed by CYP2C8 and CYP2C19, while CYP2C18 is not expressed in liver
(Pelkonen et al., 2008; Edwards et al., 1998; Shimada et al., 1994; Gray et al., 1995; Richardson et
al., 1997). CYP2C9 is a major CYP2C isoform in the human liver, and it is one of several CYP2C
genes clustered in a 500kb region on the proximal 10q24 chromosomal region (Gray et al.,
1995; Goldstein and de Morais, 1994). In Caucasian populations, the frequencies of the two
variant alleles, CYP2C9*2 and CYP2C9*3, range from 7% to 19% (Furuya et al., 1995; IngelmanSundberg et al., 1999; Miners & Birkett, 1998; Stubbins et al., 1996; Sullivan-Klose et al., 1996;
Yasar et al., 1999). CYP2C19, another member of the CYP2C enzyme family, represents
approximately 5% of the total hepatic CYPs and metabolizes drugs that are amides or weak
bases with two hydrogen bond acceptors (Pelkonen et al., 2008; Lewis, 2004; Musana & Wilke,
2005). Poor metabolizers with low CYP2C19 activity represent 3 to 5% of Caucasians and
African-Americans, and 12 to 23% of most Asian populations (Goldstein, 2001).
2.5 CYP2D subfamily
CYP2D6 represents 1 to 5% of the total CYP, and approximately 3.5 and 5-10% of the
Caucasian population are ultra-rapid and poor metabolize’s for this enzyme, respectively. The
CYP2D6 gene is clearly the most polymorphic of all known cytochrome P450s; more than 75
polymorphisms have been identified. Four alleles account for > 95% of the functional variation
observed in the general population (Pelkonen et al., 2008; Shimada et al., 1994; Musana &
Wilke, 2005; Al, Omari, A., & Murry, 2007; Ingelman-Sundberg, 2004; Zanger et al., 2004).
2.6 CYP2E subfamily
Only one gene belonging to this subfamily, namely CYP2E1, has been identified (Nelson et
al., 1996; Nelson et al., 2004). CYP2E1 is one of the most abundant hepatic CYPs, represents
15% of the total P450 and it is also expressed in lung and brain (Pelkonen et al., 2008; Raunio
et al., 1995).
2.7 CYP3A subfamily
In humans, the CYP3A subfamily contains three functional proteins, CYP3A4, CYP3A5, and
CYP3A7, and one pseudoprotein, CYP3A34. The human CYP3 family constitutes
approximately 30 % of total hepatic P450 and is estimated to mediate the metabolism of
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around 50% of prescribed drugs as well as a variety of environmental chemicals and other
xenobiotics. Because of the large number of drugs metabolized by CYP3A4, it frequently
plays a role in a number of drug-drug interactions (Pelkonen et al., 2008; Shimada et al.,
1994; Musana & Wilke, 2005; Bertz & Granneman, 1997; Domanski et al., 2001; Imaoka et al.,
1996; Rostami-Hodjegan & Tucker, 2007).
CYP3A4 is the major form of P450 expressed in human liver. It is also the major P450
expressed in human gastrointestinal tract, and intestinal metabolism of CYP3A4 substrate can
contribute significantly to first-pass elimination of orally ingested xenobiotics (Guengerich,
1995; Guengerich, 1999). X-ray crystallography studies demonstrated that CYP3A4 has a very
large and flexible active site, allowing it to oxidize either large substrates such as erythromycin
and cyclosporine or multiple smaller ligands (Scott & Halpert, 2005; Tang & Stearns, 2001).
CYP3A5 is a minor polymorphic CYP isoform in human liver in addition to the intestine
(Lin et al., 2002; Paine et al., 1997) and kidney (Haehner et al., 1996). Functional CYP3A5 is
expressed in approximately 20% of Caucasians and about 67% of African-Americans (Kuehl
et al., 2001). CYP3A5 may have a significant role in drug metabolism particularly in
populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit
CYP3A4 (Soars et al., 2006).
Expression of CYP3A7 protein is mainly confined to fetal and newborn livers, although in
rare cases CYP3A7 mRNA has been detected in adults (Hakkola et al., 2001; Kitada &
Kamataki, 1994; Schuetz et al., 1994).
3. Xenobiotic biotransformation
Xenobiotic biotransformation is the process by which lipophilic foreign compounds are
metabolized through enzymatic catalysis to hydrophilic metabolites that are eliminated
directly or after conjugation with endogenous cofactors via renal or biliary excretion. These
metabolic enzymes are divided into two groups, Phase I and Phase II enzymes (Rendic & Di
Carlo, 1997; Oesch et al., 2000).
Phase I products are not usually eliminated rapidly, but undergo a subsequent reaction in
which an endogenous substrate such as glucuronic acid, sulfuric acid, acetic acid, or an
amino acid combines with the newly established functional group to form a highly polar
conjugate to make them more easily excreted. Sulfation, glucuronidation and glutathione
conjugation are the most prevalent classes of phase II metabolism, which may occur directly
on the parent compounds that contain appropriate structural motifs, or, as is usually the
case, on functional groups added or exposed by Phase I oxidation (LeBlanc & Dauterman,
2001; Rose & Hodgson, 2004; Zamek-Gliszczynski et al., 2006).
3.1 In vitro and human-derived techniques for testing xenobiotic metabolism
In order to study the metabolism and interactions of pesticides in humans we have to rely
upon in vitro and human-derived techniques. In vitro systems have become an integral part
of drug metabolism studies as well as throughout the drug discovery process and in
academic research (Pelkonen et al., 2005; Pelkonen & Raunio, 2005; Lin & Lu, 1997). In vitro
approaches to predict human clearance have become more frequent with the increase in the
availability of human-derived materials (Skett et al., 1995). All models have certain
advantages and disadvantages, but the common advantage to these approaches is the
reduction of the complexity of the study system. However, the use of in vitro models is
always a compromise between convince and relevance (Pelkonen et al., 2005; Brandon et al.,
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
445
2003; Pelkonen & Turpeinen, 2007; Pelkonen & Turpeinen, 2007). An overview of different in
vitro models and their advantages and disadvantages are collected in Table 1.
Enzyme
sources
Availability
Advantages
Disadvantages
Contains basically all hepatic
enzymes.
Liver architecture lost.
Cofactors are necessary.
Relatively good,
from
transplantations or
commercial
sources.
Contains important ratelimiting enzymes. Inexpensive
technique. Easy storage. Study
of individual, gender-, and
species-specific
biotransformation.
Contains only CYP and
UGTs.
Requires strictly specific
substrates and inhibitors or
antibodies.
Cofactor addition
necessary.
Commercially
available
The effect of only one
The role of individual CYPs in
enzyme at a time can be
the metabolism can be easily
evaluated.
studied. Different genotypes.
Problems in extrapolation
High enzyme activities.
to HLM and in vivo.
Difficult to obtain,
relatively healthy
tissue needed.
Commercially
available
Contains the whole
complement of CYPs
cellularly integrated. The
induction effect can be
studied. Well established and
characterized. Transporters
still present and operational.
Difficult to obtain,
fresh tissue
needed.
Contains the whole
complement of CYPs and cellRequires specific
cell connections. The
techniques and well
induction, morphology and
established procedures
interindividual variation can
be studied.
Relatively good.
Commercially
available. Human
Liver
liver samples
homogenatesa
obtained under
proper ethical
permission.
Microsomesa
cDNAexpressed
individual
CYPsb
Primary
hepatocytesc,d
Liver slicese
Available upon
Immortalized request, not many
characterized cell
cell linesf
lines exist.
Non-limited source of
enzymes. Easy to culture.
Relatively stable enzyme
expression level. The
induction effect can be
studied.
Requires specific
techniques and well
established procedures.
The levels of many CYPs
decrease rapidly during
cultivation.
Cell damage during
isolation.
The expression of most
CYPs is poor.
a (Kremers, 1999); b (Rodrigues, 1999); c (Guillouzo, 1995); d (Gomez-Lechon et al., 2004); e
(Olinga et al., 1998); f (Allen et al., 2005).
Table 1. An overview of different in vitro models and their advantages and disadvantages
(modified from (Pelkonen et al., 2005; Pelkonen & Raunio, 2005; Brandon et al., 2003;
Pelkonen & Turpeinen, 2007)).
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3.2 In vitro characterization of the metabolism and metabolic interactions of
xenobiotics
The aim of in vitro characterization is to produce relevant and useful information on
metabolism and interactions to anticipate, and even to predict, what happens in man. Each
in vitro model has its own set of advantages and disadvantages as they range from simple to
more complex systems: individual enzymes, subcellular fractions, cellular systems, liver
slices and whole organ, respectively (Pelkonen et al., 2005; Pelkonen & Raunio, 2005;
Brandon et al., 2003). To understand some of the factors related to xenobiotic metabolism
that can influence the achievement of these aims, there are several important points to
consider such as (Pelkonen et al., 2005; Pelkonen & Raunio, 2005; Pelkonen & Turpeinen,
2007; Hodgson & Rose, 2005):
Determination of the metabolic stability of the compound
Identification of reactive metabolites
Evaluation of the variation between species
Identification of human CYPs and their isoforms involved in the activation or
detoxification
Evaluation of the variation between individuals
Identification of individuals and subpopulations at increased risk
Overall improvement of the process of human risk assessment
An overview of different in vitro studies for the characterization of metabolism and
metabolic interactions of xenobiotics are collected in Table 2.
In vitro test
In vitro model
Parameters
Metabolic
stability
Microsomes
Homogenates
Cells
Slices
clearance
Disappearance of the Intrinsic
parent
molecule
or Interindividual variability
appearance of (main) Interspecies differences
metabolites
Metabolite
identification
and
quantitation
Microsomes
Homogenates
Cells
Slices
Tentative identification Metabolic
routes
by (e.g.) LC/TOF-MS
Semi-quantitative
Interspecies differences
ability
of
with Relative
Identification of Microsomes
inhibitors or inhibitory enzymes to metabolize a
metabolizing
compound
antibodies
enzymes
Recombinant CYPs
Hepatocytes
Extrapolations
Prediction of effects of
various
genetic,
environmental
and
pathological
factors
Interindividual variability
Enzyme
inhibition
Inhibition of specific Potential interactions
Microsomes
Recombinant enzymes model substrate
Hepatocytes
Enzyme
induction
Cells
Slices
Permanent cell lines
Induction of CYP model Induction potential of a
activities or mRNA
substance
Table 2. In vitro studies for the characterization of the metabolism and metabolic
interactions of xenobiotics (modified from (Pelkonen et al., 2005; Pelkonen and Raunio,
2005)).
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
447
4. The contribution of CYPs to the metabolism of xenobiotics in human
4.1 CYP1A subfamily
The catalytic activities of CYP1A2 have been reviewed by Pelkonen et al. (Pelkonen et al.,
2008). CYP1A2 has a major role in the metabolism of many important chemicals such as
caffeine (Butler et al., 1989; Tassaneeyakul et al., 1992), phenacetin (Sesardic et al., 1990;
Venkatakrishnan et al., 1998), theophylline (Sarkar & Jackson, 1994; Tjia et al., 1996), clozapine
(Fang et al., 1998), melatonin (Facciolá et al., 2001; von Bahr et al., 2000), and tizanidine
(Granfors et al., 2004a; Granfors et al., 2004b). CYP1A1 is a major enzyme in the metabolism of
a number of insecticides and herbicides (Lang et al., 1997; Tang et al., 2002; Abass et al., 2010;
Abass et al., 2007c). CYP1A2 mediates herbicides (Lang et al., 1997; Abass et al., 2007c;
Nagahori et al., 2000), insecticides (Tang et al., 2002; Stresser & Kupfer, 1998; Foxenberg et al.,
2007; Mutch & Williams, 2006), and pyrethroids metabolism (Scollon et al., 2009).
4.2 CYP2A subfamily
It has been shown that CYP2A6 has a major role in the metabolism of nicotine in vitro and in
vivo (Kitagawa et al., 1999; Messina et al., 1997; Nakajima et al., 1996a; Nakajima et al., 1996b;
Yamazaki et al., 1999) and in the activation of aflatoxin B1 (Yun et al., 1991; Salonpää et al.,
1993). More substrates and inhibitors currently known to be metabolized by or to interact with
CYP2A6 in vitro and in vivo have been summarized by Pelkonen and co-workers (Pelkonen et
al., 2008; Pelkonen et al., 2000). CYP2A6 participates in the metabolism of quite a few
pesticides such as carbaryl, imidacloprid, DEET, carbosulfan and diuron (Tang et al., 2002;
Abass et al., 2010; Abass et al., 2007c; Schulz-Jander and Casida, 2002; Usmani et al., 2002).
4.3 CYP2B subfamily
CYP2B6 is known to metabolize a large number of substrates including drugs, pesticides
and environmental chemicals, many of which have been described in detail in reviews (see
e.g. (Ekins & Wrighton, 1999; Hodgson & Rose, 2007b; Turpeinen et al., 2006)). Several
clinically used drugs such as cyclophosphamide, bupropion, S-mephenytoin, diazepam,
ifosamide and efavirenz are metabolized in part by CYP2B6 (Granvil et al., 1999; Haas et al.,
2004; Huang et al., 2000; Jinno et al., 2003; Roy et al., 1999b; Roy et al., 1999a). CYP2B6
appears to activate and detoxify a number of precarcinogens (Code et al., 1997; Smith et al.,
2003). CYP2B6 plays a major role in pesticides metabolism. CYP2B6 mediates herbicides Ndealkoxylation (Coleman et al., 2000); organophosphate insecticides desulfuration
(Foxenberg et al., 2007; Mutch & Williams, 2006; Buratti et al., 2005; Leoni et al., 2008; Sams
et al., 2000; Tang et al., 2001); orghanochlorine and carbamate insecticides sulfoxidation
(Abass et al., 2010; Casabar et al., 2006; Lee et al., 2006); fungicide metalaxyl Odemethylation and lactone formation (Abass et al., 2007b).
4.4 CYP2C subfamily
CYP2C8 meditates amodiaquine N-deethylation, which is the selective marker activity,
paclitaxel 6 -hydroxylation and cerivastatin demethylation (Li et al., 2002; Rahman et al.,
1994). A few insecticides are mainly metabolized by CYP2C8 such as parathion,
deltamethrin, esfenvalerate, and -cyfluthrin (Mutch & Williams, 2006; Scollon et al., 2009;
Mutch et al., 2003; Godin et al., 2007).
CYP2C9 is responsible for the metabolism of the S-isomer of warfarin (Rettie et al.,
1992). CYP2C9 also metabolizes tolbutamide, the selective marker, glipizide, fluvastatin,
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Fungicides
phenytoin, several non-steroidal anti-inflammatory agents and many other drug groups
(Miners & Birkett, 1998; Doecke et al., 1991; Kirchheiner & Brockmoller, 2005; Rettie and
Jones, 2005). CYP2C9 is found to be involved in the metabolism of pesticides such as
pyrethroid insecticides (Scollon et al., 2009; Godin et al., 2007), as well as organophosphorus
insecticides (Leoni et al., 2008; Usmani et al., 2004).
CYP2C19 participates in the metabolism of many commonly used drugs including the
antiepileptics phenytoin and mephenytoin (Bajpai et al., 1996; Komatsu et al., 2000; Tsao et
al., 2001; Wrighton et al., 1993)(Bajpai et al. 1996, Komatsu et al. 2000, Tsao et al. 2001,
Wrighton et al. 1993), selective serotonin receptor inhibitors citalopram and sertraline
(Kobayashi et al., 1997; von Moltke et al., 2001), the psychoactive drugs amitriptyline
(Venkatakrishnan et al., 1998; Jiang et al., 2002) and diazepam, among others (Jung et al.,
1997). Among the substrates of CYP2C19 are several widely used pesticides such as the
phosphorothioate insecticides (Foxenberg et al., 2007; Mutch & Williams, 2006; Leoni et al.,
2008; Tang et al., 2001; Usmani et al., 2004; Buratti et al., 2002; Kappers et al., 2001), as well as
the pyrethroid insecticides (Scollon et al., 2009; Godin et al., 2007).
4.5 CYP2D subfamily
CYP2D6 metabolizes approximately 20 % of all commonly prescribed drugs in vivo
(Brockmöller et al. 2000). For example, CYP2D6 contributes to the metabolism of
betablockers metoprolol and timolol (Johnson & Burlew 1996, Volotinen et al. 2007) and the
psychotropic agents amitriptyline and haloperidol (Coutts et al. 1997, Fang et al. 1997, Fang
et al. 2001, Halling et al. 2008, Someya et al. 2003). Dextromethrophan O-demethylation is the
most used in vitro model reaction for CYP2D6 activity (Kronbach et al. 1987, Park et al. 1984).
Known pesticide substrates for CYP2D6 include phosphorothioate insecticides (Mutch et al.
2003, Mutch & Williams 2006, Sams et al. 2000, Usmani et al. 2004b) as well as (Johnson and
Burlew, 1996; Volotinen et al., 2007) carbamate insecticide (Tang et al., 2002). CYP2D6 is also
involved in the N-dealkylation of the atrazine and diuron herbicides (Lang et al., 1997;
Abass et al., 2007c).
4.6 CYP2E subfamily
The metabolism of very few clinically important drugs such as paracetamol, caffeine,
acetaminophen, enflurane and halothane seems to be mediated to some extent by CYP2E1 (Gu
et al., 1992; Lee et al., 1996; Raucy et al., 1993; Thummel et al., 1993). Chlorzoxazone is probably
the most used in vitro model substrate for CYP2E1 activity (Peter et al., 1990). Few pesticides
have been reported to be metabolized at least in part by human CYP2E1 such as atrazine,
carbaryl, parathion, imidacloprid and diuron (Lang et al., 1997; Tang et al., 2002; Abass et al.,
2007c; Mutch & Williams, 2006; Schulz-Jander & Casida, 2002; Mutch et al., 2003).
4.7 CYP3A subfamily
CYP3A4 participates in the metabolism of several clinically important drugs such as
triazolam, simvastatin, atorvastatin, and quinidine (Rendic & Di Carlo, 1997; Bertz &
Granneman, 1997). Detailed characteristics of several CYP3A4 substrates and inhibitors
were summarized recently by Liu et al. (Liu et al., 2007). The known pesticides mainly
metabolized by CYP3A4 belong to several chemical groups such as, carbamate,
phosphorothioate, chlorinated cyclodiene and neonicotinoid insecticides (Tang et al., 2002;
Abass et al., 2010; Mutch & Williams, 2006; Schulz-Jander & Casida, 2002; Buratti et al., 2005;
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
449
Sams et al., 2000; Tang et al., 2001; Casabar et al., 2006; Lee et al., 2006; Mutch et al., 2003;
Usmani et al., 2004; Buratti et al., 2002; Buratti et al., 2003; Buratti & Testai, 2007; Butler and
Murray, 1997), herbicides (Abass et al., 2007c; Coleman et al., 2000), fungicides (Abass et al.,
2007b; Abass et al., 2009; Mazur et al., 2007), and organotin biocide (Ohhira et al., 2006).
CYP3A5 mediates midazolam, alprazolam and mifepristone metabolism (Christopher
Gorski et al., 1994; Galetin et al., 2004; Hirota et al., 2001; Huang et al., 2004; Khan et al.,
2002; Williams et al., 2002). Alprazolam has been suggested as a selective probe for CYP3A5
(Galetin et al., 2004). The metabolism of a number of organophoshate and pyrethroid
insecticides has been reported to be mediated by CYP3A5 (Mutch & Williams, 2006; Mutch
et al., 2003; Godin et al., 2007).
CYP3A7 has similar catalytic properties compared with other CYP3A enzymes, including
testosterone 6 -hydroxylation (Kitada et al., 1985; Kitada et al., 1987; Kitada et al., 1991).
5. The impact of modern analytical techniques in xenobiotics metabolism
5.1 Mass spectrometric methods in metabolism studies.
Traditionally metabolism studies were performed using gas chromatography-mass
spectrometry (GC-MS). Because metabolites are usually polar molecules with high
molecular masses, they have to be derivatised before measurement. After derivatisation the
measured analyte is not anymore original metabolite but a less polar compound which is
possible to vaporise and use in GC. Derivatisation can cause errors to the measurements and
it is usually the most time and labour demanding phase which causes extra costs (Sheehan,
2002). In metabolism studies biggest problem of GC-MS is its lower sensitivity compared
with modern mass spectrometric methods. Nevertheless GC-MS is still a useful method also
in metabolism studies especially with thermally stable volatile compounds.
Nowadays the primary method used in metabolism studies is liquid chromatogram-mass
spectrometry (LC-MS). Liquid chromatography is an old technique to separate polar
compounds in liquid phase. However, it took quite a long time to develop a reliable technique
to connect LC to the mass spectrometer, because the eluent solvent has to be vaporized before
actual MS measurements which are performed in high vacuum. However after the
introduction of electrospray ionisation (ESI) development has been very rapid during last 20
years and the performance of instruments has steadily improved. Usually ESI is the best choice
for the ionization of polar metabolites but there are also other common ionisation methods like
APCI (atmospheric pressure chemical ionisation) and APPI (atmospheric pressure photoionisation), which can be used to ionise less polar compounds. ESI can be run either in positive
or negative modes and the best choice is dependent on the specific analyte. During ionisation
hydrogen is either combined with the analyte to produce [M+H]+ ion or broken away to
produce [M-H]— ion, which can be accelerated within electric field. In the same time usually
also other adducts, like sodium and potassium adducts, are formed. Sometimes other adducts
can cause problems or decrease the sensitivity of the method. In addition other compounds
that elute at same time from LC flow can reduce or block totally the ionisation of the analyte
and cause errors to the measurements. This phenomenon is called ion suppression and it is
quite common in ESI (Jessome & Volmer, 2006).
The most useful sample handling procedure to be used in metabolism studies with LC-MS is
protein precipitation. It is performed easily by addition of organic solvent, either methanol
or acetonitrile, to the samples. Samples are mixed and centrifuged to get clear supernatant.
Usually after protein precipitation samples are clean enough to be analyzed directly, but
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Fungicides
also other sample handling method may be needed with samples containing a lot of lipids
or salts (Rossi, 2002). Different extraction methods, like SPE (solid phase extraction) or
liquid-liquid extraction are then a better choice. However they are more expensive and time
consuming methods.
Already HPLC (high performance liquid chromatography) is able to separate metabolites
directly without any modifications. Compared to the GC, the resolution of HPLC is quite
poor. Because mass spectrometric methods can measure compounds coming to the
instrument at the same time, this has not been so big a problem. During the last five years
liquid chromatography has improved considerably after introduction of ultra performance
liquid chromatography (UPLC). UPLC instruments can work in higher operation pressures
(up to 15.000 psi) which makes possible to use smaller particles and diameters in columns
and to improve resolution, speed and sensitivity of the method. A typical run in UPLC can
be just 5 minutes to analyse several different compounds.
Mass spectrometry is a superior method in the metabolism studies because of its high
sensitivity. Although mass spectrometry is usually understood as one concept, it actually
consist of several different types of instruments and techniques. Different types of
instruments have specific advantages and consequently each individual type suits best for
certain kind(s) of measurements. In the identification of metabolites time of flight mass
spectrometry (TOF) is the best option. It can detect all ionized compounds simultaneously
which improve the sensitivity compared to scanning instruments (Fountain, 2002). With
help of the TOF instruments accurate mass of the analyte (±5 ppm) can be measured and
elemental composition can be calculated. Modern instruments can easily reach 1 ppm mass
accuracy and use isotope patterns of analytes to solve the right elemental composition with
few potential possibilities. This kind of identification can be used to find different
metabolites in samples, because masses of potential metabolites can usually be calculated
before measurements. There are also softwares, such as Metabolynx (Waters Corp., Milford,
MA, USA), which can search potential metabolites automatically from mass chromatograms
and help a lot in data processing.
Additional structural information can be achieved with help of Q-TOF or triple quadrupole
instruments. Measured analytes can be decomposed by collision with gas molecules (CID,
collision induced dissociation) to produce fragment ions. In Q-TOF instruments accurate
mass of fragment ions can be also measured to resolve molecular masses of fragments. In
most cases fragmentation produces information about location of possible
biotransformations. Because fragmentation is compound-specific, fragments can be used for
identification purposes if they are known from previous measurements. However
fragmentation is not as universal as in EI-ionisation (electron ionisation) of GC-MS
instruments because it is partly instrument specific. With ion trap instruments even
produced fragment ion can be selected and collided again to produce new smaller fragment
ions. To resolve the structure of a metabolite completely other methods like nuclear
magnetic resonance (NMR) or x-ray crystallography are usually required.
Knowledge about fragmentation of the analyte is useful also in quantitative measurements.
Quantifications are usually performed in triple quadrupole instruments, where
fragmentation can be used to increase selectivity of the measurements. The mode of the
measurement is called multiple reaction monitoring (MRM), because several compounds
can be measured simultaneously. In triple quadrupole instruments the first quadrupole
selects the measured analyte, the second one decomposes it and the third passes the formed
fragment to the detector. Because fragmentation is specific to every analyte, only right one is
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
451
measured even if compounds with the same molecular mass come to the instrument at the
same time. This kind of high selectivity makes also possible to measure several compounds
at the same time even when they are not separated in liquid chromatography. In triple
quadrupole instruments dynamic range is usually at least 5 orders of magnitude what is
enough for quantification purposes. Earlier TOF instruments were saturated at quite low
concentrations and could not be used for quantification purposes. Modern TOF instruments
however can be used for quantification at least to a certain extent.
Newest technological addition to mass spectrometry is ion mobility. Ion mobility is a small gas
filled drift tube in instruments, which ions travel through within electric field. Drift tube will
separate compounds based on their shape and size in addition to mass and charge as in
conventional instruments. Ion mobility is quite an old technique but just recently it has been
combined with commercial mass spectrometers like Synapt HDMS (Waters Corp., Milford,
MA, USA) (Kanu et al., 2008). Ion mobility can be used to clean important analytes from
sample matrix and to separate very similar compounds like isomers from each other. Because
technique is so new, its real practical significance in metabolism studies is still unclear.
Figure 3 presents a practical example about mass spectrometric measurements of the
pesticide profenofos and its metabolite hydroxypropylprofenofos (Abass et al., 2007a).
Accurate mass measurements were performed by Micromass LCT-TOF (Micromass,
Altrincham, UK) using leucine enkephalin ([M+H]+ at m/z 556.2771) as a lock mass
compound. Error in accurate mass measurements of hydroxypropylprofenofos was 4.3
mDa. Fragmentations of hydroxypropylprofenofos were determined by Micromass Quattro
II triple quadrupole instruments. In the first fragmentation hydroxypropylprofenofos loses
ethanol to produce fragment of m/z = 343 Da. In the second step propanol is released to
produce fragment of 285 Da. Difference in molecular masses of these two fragments
indicates that hydroxylation has to be located in S-propyl moiety of the metabolite. Finally
quantifications were performed in multiple reaction monitoring mode (MRM) of triple
quadrupole instruments. Quadrupole 1 passes only hydroxypropylprofenofos (molecular
mass 389 Da) or compounds with the same molecular mass. After quadrupole 1
hydroxypropylprofenofos will fragment in collision cell with help of argon gas and collision
energy (CE= 20 eV) to produce a specific fragment of m/z 343 Da. In the final step only
fragment 343 will pass quadrupole 3 and its amount is determined in the detector of the
instrument. After calibration of the instrument with reference standards, the real amount of
hydroxypropylprofenofos can be determined.
5.2 Nuclear Magnetic Resonance spectrometry in the metabolism studies.
Nuclear Magnetic Resonance spectroscopy (NMR) is a powerful analytical tool in studies of
solid, gaseous and liquid samples. The versatility of the technique and the long relaxation
times of the nuclear spins allow for probing various different properties of the samples. An
even normal, simple one-dimensional spectrum contains valuable information about the
sample concentration, electron distributions of the molecule, spatial proximities of different
chemical sites and electrostatic connectivities between different nuclei of the molecule. The
full potential of NMR can be unleashed by going into higher dimensional NMR
spectroscopy. In typical two-, or three-dimensional NMR spectra one can probe spatial
proximities of various nuclei, do diffusion separated spectroscopy, probe for heteronuclear
connectivities multiple bonds away, or characterize intermolecular dipolar interactions at
the protein-ligand complex interface.
452
Fungicides
Accurate mass measurement of hydroxypropylprofenofos by time-of-flight mass
spectrometer:
Cl
Cl
O
O
O
O
P
O
S
O
P
S
Br
Br
n-Pr-OH
LC/MS-ESI+, MH+=373Da
Accurate mass: 372.9477, Calc. mass: 372.9430
Profenofos
O-(4-bromo-2-chlorophenyl)-O-ethyl-S-propyl
phosphorothioate
LC/MS-ESI+, MH+=389Da
Accurate mass: 388.9422, Calc. mass: 388.9379
Hydroxypropylprofenofos
O-(4-bromo-2-chlorophenyl)-O-ethyl-S-hydroxy
-propyl phosphorothioate
The fragmentation of hydroxypropylprofenofos by triple quadrupole mass spectrometer:
H
O
O
Cl
Cl
+
O
O
P
P
+
O
Cl
O
S
S
Br
Br
H
OH
P
+
O
S
Br
OH
m/z = 389 Da
m/z = 285 Da
m/z = 343 Da
The quantification of hydroxypropylprofenofos by triple quadrupole mass spectrometer:
Quadrupole 1
Collision cell
Quadrupole 3
Ion
source
m/z = 389 Da
Ar
Ar
Ar
m/z = 343 Da
CE = 20 eV
Detector
CE = 20 eV
Fig. 3. Mass spectrometric measurements, accurate mass, fragmentations and
quantifications, performed to study hydroxylation of profenofos in human hepatic
subcellular fractions.
The biggest drawback of the NMR spectroscopy is its inherently low sensitivity, because the
observed signal arises from the population difference of spin states. This population
difference follows Boltzmann distribution and is quite low even at reasonably high magnetic
fields used at modern NMR spectrometers. As the experiments are performed close to room
or physiological temperature, there is only population difference of about 10 spins for every
million spins in the sample.
Two recent sensitivity enhancing methods are cryogenic cooling of the probe-head
electronics and miniaturization of the sample size. Equipment for both of these have been
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
453
commercially available now for several years and when combined the resulting cryomicroprobe would give up to 15 fold increase in sensitivity compared to regular room
temperature probe head (Kovacs et al., 2005). Several articles have recently been published
where full NMR analyses of complex natural products have been made using only
nanomoles of material (would be equal to 1 µg if molecular mass is 1000) (Dalisay &
Molinski, 2009; Choi et al., 2010a; Choi et al., 2010b; Djukovic et al., 2008).
If one wishes the acquisition parameters of NMR experiment can be set to provide
quantitative spectrum. Typically the delay between individual transitions needs to be
lengthened to allow full relaxation of spins before next transition. In simplest form the
integral values of the individual resonances in spectrum give information of how many
equivalent spins are present. Whenever there are modifications in chemical structure the
change in integral provides valuables information on the chemical site of the modification
(Holzgrabe, 2010).
The Chemical shift is a sensitive measure of the electronic surrounding of individual nuclei
of a molecule. Even smallest changes in the chemical structure can cause peaks to resonate at
slightly different frequency at the chemical shift range. Addition of the electronegative
substituents to the molecule changes the chemical shifts of the resonances of the nearby
nuclei. In favorable case the change in chemical shift can be observed for several nucleus
many bonds away from the origin of modification site.
The signal fine structure, the splittings caused by spin-spin couplings, provides additional
sensitive measure of the topology of the nucleus in the molecule. Change in number of
nearby nucleus or even just a conformational change can be detected as a change observed
coupling pattern caused by the spin-spin coupling.
In metabolic studies NMR spectroscopy is best utilized when used as a complementary
technique to the mass spectrometric techniques. For instance the position isomerism studies
are often quite tricky or even impossible to solve by mass spectroscopy e.g. what is the
substitution pattern of the aromatic ring or which carbon of the aliphatic chain was
hydroxylated. These questions can occasionally be answered in minutes by single 1H NMR
spectrum. Of course more challenging structural questions take longer and might require
acquisition of several multi-dimensional data sets.
For the illustration of the position isomerism detection powers of NMR spectroscopy several
simulated NMR spectra of the profenofos and the hydroxypropylprofenofos are displayed
in figure 4. On the left are the aromatic signals of the profenofos and the corresponding
spectrum if the bromine was in ortho position to the chlorine. The difference in signal
positions and splitting patterns is clear. On the right are spectra of the propyl moiety of the
profenofos and the hydroxypropyl moiety of the hydroxypropylprofenofos where the
hydroxylation has occurred on terminal carbon 3 or in carbon 2.
6. Conclusion
The cytochrome P450 (CYP) superfamily comprises a broad class of phase I oxidative
enzymes that catalyze many hepatic metabolic processes. Human CYPs have broad
substrate specificity and enzymes in families 1-3 function mostly in the metabolism of a
wide variety of xenobiotics. In human liver, CYP3A4 is found in the highest abundance and
it metabolizes the greatest number of drugs and a very large number of other xenobiotics.
CYP enzymes in humans as well as in other species have been intensively studied during
recent years. It is now possible to characterize metabolism, metabolic interactions and to
454
Fungicides
Fig. 4. An example of the effect of small changes in molecular structure to the outlook of 1H
NMR spectrum illustrated by 5 simulated NMR spectra. The values used for chemical shifts
and coupling constants are only approximate and are presented for illustration purposes
only. The values of signal integration are displayed below the frequency scale.
determine which P450 is involved in the metabolism of a certain xenobiotic by different in
vitro approaches. The toxicity of many types of pesticides is mediated by enzymatic
biotransformation reactions in the body. Recently, a number of papers have been published
on the activity of human P450s involved in the metabolism of pesticides and these activities
may result in activation and/or detoxification reactions.
The aim of in vitro characterization is to produce relevant and useful information on
metabolism and interactions to predict what happens in vivo in human. To understand
some of the factors related to xenobiotics, including pesticides, metabolism that can
influence the achievement of these aims, there are several important points to consider such
as metabolic stability, metabolic routes and fractional proportions, metabolizing enzymes
Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human?
455
and potential interactions. In this review we described the human xenobiotic- metabolizing
enzymes CYPs system; briefly illustrate in vitro human-derived techniques for studying
xenobiotic metabolism and in vitro characterization of metabolic characteristics; review the
role of CYPs in the metabolism of xenobiotics, including drugs and pesticides, in human in
vitro; and finally describe the impact of modern analytical techniques in xenobiotics
metabolism.
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