Metabolism of Pesticides by Human Cytochrome P450 Enzymes In Vitro – A Survey

Cytochrome P450 enzymes (CYPs) are active in the metabolism of wide variety of xenobiotics. The investigation of the contributions of human CYPs in pesticides metabolism, especially insecticides, is still growing. One of the background tools to facilitate this task is by sorting the contribution of each human CYP isoform in the metabolism of pesticides. This paper attempts to provide a comprehensive literature survey on the role of human hepatic CYPs such as human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7 in pesticides biotransformation in vitro as well as to sort the reactions mediated. Based on relevant publications identified by searching databases from 1995 through 2011, more than 400 metabolic reactions were reported to be mediated at least in part by human CYPs in vitro. Some information on older papers was obtained from previous literature surveys compiled by Hodgson 2001 & 2003. Finally, we give brief insight into potential modulations and consequences of human CYP genes – pesticides interactions.


Introduction
Cytochrome P450 enzymes (CYPs) are active in the metabolism of wide variety of xenobiotics. The investigation of the contributions of human CYPs in pesticides metabolism, especially insecticides, is still growing. One of the background tools to facilitate this task is by sorting the contribution of each human CYP isoform in the metabolism of pesticides. This paper attempts to provide a comprehensive literature survey on the role of human hepatic CYPs such as human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7 in pesticides biotransformation in vitro as well as to sort the reactions mediated. Based on relevant publications identified by searching databases from 1995 through 2011, more than 400 metabolic reactions were reported to be mediated at least in part by human CYPs in vitro. Some information on older papers was obtained from previous literature surveys compiled by Hodgson & 2003 Finally, we give brief insight into potential modulations and consequences of human CYP genes -pesticides interactions.

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 and Di Carlo, 1997;Oesch et al. 2000). Phase I reactions are mediated primarily by cytochrome P450 family of enzymes, but other enzymes (e.g. flavin monooxygenases, peroxidases, amine oxidases, dehydrogenases, xanthine oxidases) also catalyze oxidation of certain functional groups. In addition to the oxidative reactions there are different types of 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 existing or newly added or exposed functional group to form a highly polar conjugate to make them more easily excreted (LeBlanc and Dauterman, 2001;Rose and Hodgson, 2004;Zamek-Gliszczynski et al. 2006). Fig. 2. Schematic description of the two main phases of drug metabolism. In general, a parent compound is converted into an intermediate metabolite which is then conjugated, but metabolism may involve only one of these reactions. Some metabolites are more toxic than the parent compound (Ahokas and Pelkonen, 2007;Liska et al. 2006). Microsomes have many advantages including easy adaptation to higher throughput assays, easy preparation and use, good stability during storage, high CYP concentration and high rate of metabolite turnover. Brandon et al. 2003;Ekins et al. 1999;Ekins et al. 2000;.

Function
CYP oxidation reactions involve a complex series of steps. The initial step involves the binding of a substrate to oxidized CYP, followed by a one-electron reduction catalyzed by NADPH cytochrome P450 reductase to form a reduced cytochrome-substrate complex. The next several steps involve interaction with molecular oxygen, the acceptance of the second electron from NADPH cytochrome b5 reductase, followed by subsequent release of water and the oxygenated product of the reaction. This reaction sequence results in the addition of one oxygen atom to the substrate, while the other atom is reduced to water (Parkinson, 2001;Rose and Hodgson, 2004;Guengerich, 2001) (Sohl et al. 2008) (Sohl et al. J. Biol. Chem. 2008).

In vitro approaches
In vitro approaches to characterize metabolic fate for human clearance predication have become more frequent with the increase in the availability of human-derived materials. All www.intechopen.com models have certain advantages and disadvantages, but the common advantage to these approaches is the reduction of the complexity of the study system. In vitro model range from simple to more complex systems: individual enzymes, subcellular fractions, cellular systems, liver slices and whole organ, respectively. However, the use of in vitro models is always a compromise between convenience and relevance. Different in vitro models and their advantages and disadvantages have been described previously Brandon et al. 2003;Pelkonen and Turpeinen, 2007).

Identification of the individual CYP enzyme(s) involved in the metabolism of a xenobiotic
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 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 and finally overall improvement of the process of human risk assessment. Basically the identification of the individual CYP enzyme(s) involved in the metabolism of a xenobiotic is necessary for in vitro -in vivo extrapolation and prediction if the results of the metabolic stability and metabolic routes in human in vitro systems indicate that CYP enzymes contribute significantly to the metabolism of a xenobiotic. Due to the broad substrate specificity of CYP enzymes, it is possible for more than one enzyme to be involved in the metabolism of a single compound. In vitro methods have been established to determine which CYP isoform(s) is (are) involved in the metabolism of a xenobiotic . The identification could be achieved by different approaches such as cDNA-expressed enzymes, correlation studies, inhibition studies with CYP-selective chemical inhibitors and specific antibodies and inhibition of CYP enzymes.

cDNA-expressed enzymes
The availability of a full panel of recombinant enzymes covering the major human liver CYPs allows a direct approach for assaying the metabolism of a compound by incubation with the isolated isoforms. This can be done by following substrate consumption or product formation by each isoform using the same analytical methods as for human liver microsomes-based assays (Reponen et al. 2010). The biotransformation of a xenobiotic by a single CYP does not necessarily mean its participation in the reaction in vivo. The relative roles of individual CYPs cannot be quantitatively estimated using this approach due to the interindividual variation in the levels of individual active CYPs in the liver (Guengerich, 1999;. However, cDNA-expressed CYPs are well suited for isozyme identification in a high-throughput screening format (White, 2000). The relative importance of individual isoform to in vivo clearance is dependent upon the relative abundance of each isoform. When taking into account the average composition of human hepatic CYPs, an approximate prediction of the participation of any CYP enzyme in the whole liver activity can be achieved (Rodrigues, 1999;Rostami-Hodjegan and Tucker, 2007).

Correlation studies
Using a bank of "phenotyped" liver microsomes, correlation analysis could be performed. Correlation analysis involves measuring the rate of xenobiotic metabolism by several liver samples from individual humans and correlating reaction rates with the level of activity of the individual CYP enzymes in the same microsomal samples. If there are a sufficient number of individual samples (at least ten), the correlation plot would give the information needed for the evaluation of the participating CYPs. The higher the correlation between the activities, the larger the probability that the respective CYP enzyme is responsible for the metabolism of the xenobiotic. Another approach is to correlate the levels of an individual CYP determined by Western blot analysis against the metabolic activity (Beaune et al. 1986;Brandon et al. 2003;Berthou et al. 1994;Jacolot et al. 1991;Wolkers et al. 1998).

Inhibition studies with CYP-selective chemical inhibitors and specific antibodies
Pooled human liver microsomes or individual liver microsomal samples should be used to examine the effect of CYP-selective chemical inhibitors or selective inhibitory antibodies. Antibody inhibition involves an evaluation of the effects of inhibitory antibodies against selective CYP enzymes on the metabolism of a xenobiotic in human liver microsomes. Chemical inhibition involves an evaluation of the effects of known CYP enzyme inhibitors on the metabolism of a xenobiotic. Several compounds have been characterized for their inhibitory potency against different CYPs; for example, furafylline is perhaps the most potent and selective inhibitor of CYP1A2, tranylcypromine of CYP2A6, thiotepa and ticlopidine of CYP2B6, trimethoprim and sulfaphenazole are selective inhibitors of CYP2C8 and CYP2C9, respectively, fluconazole may be used for CYP2C19, quinidine is a commonly used in vitro diagnostic inhibitor of CYP2D6 activity, pyridine and disulfiram of CYP2E1, and ketoconazole and itraconazole are among many potent and relatively selective inhibitors of CYP3A4 often used in vitro and in vivo as diagnostic inhibitors (Rendic and Di Carlo, 1997;Bourrie et al. 1996;Clarke et al. 1994;Nebert and Russell, 2002;Pelkonen et al. 2008;Schmider et al. 1995;Sesardic et al. 1990).

Inhibition of CYP enzymes
Testing the inhibitory interactions of a xenobiotic on CYP-specific model activity in human liver microsomes in vitro provides information about the affinity of the compound for CYP enzymes . The type of CYP inhibition can be either irreversible (mechanism-based inhibition) or reversible. Irreversible inhibition requires biotransformation of the inhibitor, while reversible inhibition can take place directly, without metabolism. Reversible inhibition is the most common type of enzyme inhibition and can be further divided into competitive, noncompetitive, uncompetitive, and mixedtype inhibition (Pelkonen et al. 2008). The inhibitory interactions of a xenobiotic on CYP enzymes can be tested by co-incubating a series of dilutions of a xenobiotic with a reaction mixture containing single or multiple substrates. In the single substrate assay, traditionally CYP interaction studies are performed using specific assays for each CYP isoform. A decrease in probe metabolite formation produced by inhibition is usually analyzed by LC-UV, LC-MS or fluorometry. In the cocktail assay, several CYP-selective probes are incubated with human liver microsomes and analyzed by LC-MS-MS (Tolonen et al. 2007;Turpeinen et al. 2006;Turpeinen et al. 2005;Tolonen et al. 2005).

Pesticides reported to be metabolized at least in part by certain human cytochrome P450
During the recent years, a large number of papers have been published on the activities of human CYPs involved in the metabolism of pesticides. Human CYPs involved in metabolism of pesticides and related compounds were listed and updated previously several years ago by & 20032003). Abbreviations used in the coming tables are listed in table 1. The updated human CYPs and their isoforms catalyzing pesticides biotransformation in addition to reactions detection methods are listed below in tables containing the primary CYP-specific information (Tables 2 to 13). Additional summary table contains information classified according to individual metabolic reactions and chemical classes of pesticides (Table 14).  Table 9. Pesticides reported to be metabolized at least in part by human CYP2D6.  Table 13. Pesticides reported to be metabolized at least in part by human CYP3A7.

Induction of CYP enzymes
Induction is defined as an increase in enzyme activity associated with an increase in intracellular enzyme concentration. CYP-pesticides interactions involve either induction or inhibition of metabolizing enzymes. Many induction studies have been conducted in vitro using primary human hepatocytes, human hepatoma cell lines or cell lines derived from other human tissues (Dierickx, 1999;Delescluse et al. 2001;Coumoul et al. 2002;Sanderson et al. 2002;Wyde et al. 2003;Lemaire et al. 2004). Primary culture of hepatocyte maintain whole cell metabolism since transporters and both phase I and phase II enzymes are present. Likewise, HepaRG cells express a large panel of liver-specific genes including several CYP enzymes, which is in contrast to HepG2 cell lines. In addition to P450 enzymes, HepaRG cells have a stable expression of phase II enzymes, transporters and nuclear transcription factors over a time period of six weeks in culture (Aninat et al. 2006;Anthérieu et al. 2010;Kanebratt and Andersson, 2008;Turpeinen et al. 2009). Both immunoblotting and reverse transcription polymerase chain reaction (RT-PCR) techniques have been applied to examine the pesticide-CYP induction (Wyde et al. 2003;Lemaire et al. 2004;Das et al. 2006;Sun et al. 2005;Johri et al. 2007;Barber et al. 2007). However, problems in tissue availability, interindividual differences, reproducibility and ethical issues preclude the efficient large-scale use of human primary hepatocytes for induction screening. One important factor regulating the expression of drug metabolising enzymes is induction by a diverse group of endogenous and exogenous substances that bind to the nuclear receptors pregnane X receptor (PXR) or constitutive androstane receptor (CAR), thereby causing significant up-regulation of gene transcription (Pelkonen et al. 2008;Handschin and Meyer, 2003). Therefore, the development of mechanism-based test systems for induction screening, based for example on in vitro pregnane X receptor/constitutive androstane receptor activation, is currently very active, and some test systems are in use as a first step for the identification of potential inducers . Whereas the acute effects of exposure to high doses of pesticides are well known, the longterm effects of lower exposure levels remain controversial. The ability of chemicals to induce metabolic enzymes, including cytochrome P450 (CYP), has long been considered as one of the most sensitive biochemical cellular responses to toxic insult (Gonzalez et al. 1993;Denison and Whitlock Jr., 1995), since it often occurs at much lower doses of the chemical than those known to cause lethal or overtly toxic effects. Assessment of inducibility of xenobiotic-metabolising enzymes by pesticides is vital for health risk assessment. Numerous pesticides are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. Several articles on CYP gene inducibility by pesticides and other chemicals used in agriculture and public health have been published (Abass et al. 2009) and a review article dealing with CYP gene modulation by pesticides is needed.

Acknowledgements
This work was supported by a grant from KONE foundation -Finland.