Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines
Introduction
Variability in drug metabolism and drug transport plays an important role in human drug toxicology and therapeutic efficacy (Wormhoudt et al., 1999). An important aspect in the development of new drugs is therefore the elucidation of drug metabolism pathways, assessment of the pharmacological and toxicological activity of formed metabolites compared to the parent drug, identification of the metabolic enzymes involved in the biotransformation and the elucidation of the role of drug transport in the uptake and excretion of a drug.
Biotransformation pathways can be divided into two categories; phase I (oxidation, reduction and hydrolysis) and phase II (conjugation) reactions (Derelanko and Hollinger, 1995, Crommentuyn et al., 1998). The cytochrome P450 (CYP) superfamily is the largest and most important group of the phase I enzymes and the CYP3A, CYP2D and CYP2C subfamilies are responsible for, respectively, 50%, 25 and 20% of the biotransformation of all drugs (Smith and Jones, 1991, Wrighton and Stevens, 1992, Lu, 1996). Major enzymes involved in phase II reactions are uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT) and glutathione-S-transferase (GST) (Lu, 1996, Range et al., 1996, Ritter, 2000).
The drug transporters of the ATP binding cassette (ABC)-containing family of proteins have a well-established effect on the pharmacokinetics of many clinically relevant drugs. These transporters influence the oral bioavailability and the hepatobiliary, intestinal and urinary excretion of drugs and their metabolites. The most important members of the ABC-family are P-glycoprotein (P-gp, MDR1), multidrug resistance protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) (reviewed by Schinkel and Jonker, 2003).
Several strategies have been developed to study human drug metabolism and active transport of drugs in vitro, like the use of transformed cell lines (reviewed by Brandon et al., 2003, Plant, 2004, Varma et al., 2003, Braun et al., 2000). Unfortunately, preclincial model systems used for assessment of human drug metabolism and drug transport poorly predict biotransformation and elimination in man. A number of factors may explain this discrepancy. Expression of drug-metabolizing enzymes and drug transporters in transformed cell lines is often low and variable, as in the often applied HepG2 human hepatoma cell system (Knasmuller et al., 1998, Brandon et al., 2003, Wilkening and Bader, 2003). Furthermore, genetic variability in these enzyme and transport systems in cell lines is poorly reported.
The genetic component in the inter-individual variability in enzyme activity has been estimated to be high (Wormhoudt et al., 1999). In clinical studies, it is already shown that genetic polymorphisms in biotransformation enzymes and drug transporters can have a large impact on adverse drug reactions and the therapeutic efficacy of drugs like warfarin, several psychoactive drugs and anti-cancer drugs, e.g., irinotecan (Sekine and Saijo, 2001, Scordo et al., 2002, Toffoli et al., 2003). However, in several often used cell line models to study drug metabolism and transport the occurrence of genetic polymorphisms has never been studied, despite the fact that these polymorphisms could influence the outcome of preclinical biotransformation and transport studies of drugs performed with these cell lines. The aim of this study was therefore to screen four often used transformed cell lines in biotransformation and drug transport studies, HepG2, IGROV-1, CaCo-2 and LS180 cells, for polymorphisms in the main CYPs, phase II enzymes and drug transporters.
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Materials
RPMI-1640 medium (with l-glutamine and 25 mM HEPES), heat-inactivated fetal calf serum, penicillin/streptomycin and Hanks' balanced salt solution (pH 7.4) were all obtained from Gibco BRL (Breda, The Netherlands). Primers and DNAzol were purchased from Invitrogen Life Technologies (Paisley, UK) or Metabion (Planegg-Martinsried, Germany) and the restriction enzymes BcgI, BsaI, BstEII, BstUI, DdeI, NciI, NlaIII, Tsp509 I and XcmI were provided by New England BioLabs (Hitchin, UK). The other
Genotyping of cytochrome P450 enzymes
The results of the CYP genotyping for all four cell lines are shown in Table 2. To compare the observed polymorphisms with in vivo data, the occurrence in Caucasians is also mentioned, as is the effect of the polymorphism on the activity of the concerning enzyme. The cell lines were all found to be homozygous mutant for the CYP3A5*3 polymorphism, which is a common genetic polymorphism in Caucasians (around 90%) and is known to have no enzymatic activity (Peng et al., 2003). All four cell lines
Discussion
In vitro cell models are frequently used in preclinical research of novel compounds and can be used to elucidate the biotransformation route of new drugs. However, often results obtained in vitro poorly correlate with biotransformation and elimination routes observed in man. The aim of our study was to characterize the expression of genetic polymorphisms in drug-metabolizing and ABC-drug transport systems in four different cell lines, a hepatoma cell line (Hep G2), an ovarian carcinoma
Acknowledgment
This work was supported with a grant from the Nijbakker-Morra Foundation, The Netherlands.
References (82)
- et al.
CaCo-2 monolayers in experimental and theoretical predications of drug transport
Adv. Drug. Deliv. Rev.
(2001) - et al.
CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6α-hydroxylase activity in human liver microsomes
Biochem. Pharmacol.
(2002) - et al.
Peroxisome proliferator-activated receptor α induces hepatic expression of the human bile acid glucuronidation UDP-glucuronosyltransferase 2B4 enzyme
J. Biol. Chem.
(2003) - et al.
An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons
Toxicol. Appl. Pharmacol.
(2003) - et al.
Cell cultures as tools in biopharmacy
Eur. J. Pharm. Sci.
(2000) - et al.
In-vitro metabolism of anti-cancer drugs, methods and applications: paclitaxel, docetaxel, tamoxifen and ifosfamide
Cancer Treat. Rev.
(1998) - et al.
Genetic tests which identify the principal defects in CYP2C19 responsible for the polymorphism in mephenytoin metabolism
Methods Enzymol.
(1996) - et al.
The significance of the homozygous CYP2A6 deletion on nicotine metabolism: a new genotyping method of CYP2A6 using a single PCR-RFLP
Biochem. Biophys. Res. Commun.
(1999) - et al.
CYP2A6*6, a novel polymorphism in cytochrome P450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymatic activity
J. Biol. Chem.
(2001) - et al.
Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens
Mutat. Res.
(1998)
Differences in N-acetylation genotypes between Caucasians and Black South Africans: implications for cancer prevention
Cancer Detec. Prev.
Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin
Biochem. Biophys. Res. Commun.
Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method
Toxicology
Strategies for using in vitro screens in drug metabolism
Drug Discovery Today
Roles of glucuronidation and UDP-glucuronosyltransferase in xenobiotic bioactivation reactions
Chem.-Biol. Interact.
Genotyping of cytochrome P450 2D6*3 and *4 mutations using conventional PCR
Clin. Chim. Acta
Mammalian drug efflux transporters of the Atp binding cassette (ABC) family: an overview
Adv. Drug Delivery Rev.
Polymorphisms of metabolizing enzymes and transporter proteins involved in the clearance of anticancer agents
Ann. Oncol.
Genetic polymorphism of CYP1A1, GSTM1 and GSTT1 genes in Indian oral cancer
Oral Oncol.
P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement
Pharm. Res.
Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression: characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1
Mol. Pharmacol.
Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases
Proc. Natl. Acad. Sci. U.S.A.
Genetic polymorphism of UDP-glucuronosyltransferase 2B7 (UGT2B7) at amino acid 268: ethnic diversity of alleles and potential clinical significance
Pharmacogenetics
Re-investigation of the concordance of human NAT phenotypes and genotypes
Arch. Toxicol.
Detection of SNPs in the ABCG2 gene in a Dutch population
Am. J. Pharmacogenomics
The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome
N. Engl. J. Med.
A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility
Cancer Res.
Arylamine N-acetyltransferase activity in man
Drug Metab. Rev.
Detection of three genetic polymorphisms in the 5′-flanking region and intron 1 of human CYP1A2 in the Japanese population
Jpn. J. Cancer Res.
Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells
Biosci., Biotechnol., Biochem.
Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions?
Biochem. Pharmacol.
Development of CaCo-2 cells expressing high levels of cDNA-derived cytochrome P4503A4
Pharm. Res.
A novel functional polymorphism in the uridine diphosphate-glucuronosyltransferase 2B7 promotor with significant impact on promotor activity
Clin. Pharmacol. Ther.
Quality of NAT2 genotyping with restriction fragment length polymorphism using DNA isolated from frozen urine
Cancer Epidemiol., Biomark. Prev.
Expression of drug metabolizing enzymes in human HepG2 hepatoma cells
Cellular Molec. Aspects Cirrhosis
Detection of polyol accumulation in a new ovarium carcinoma cell line, CABA I: a 1H NMR study
Br. J. Cancer
Pharmacogenetics of soluble sulfotransferases (SULTs)
Naunyn-Schmiedeberg's Arch. Pharmacol.
Mixed function oxidase and UDP-glucuronosyltransferase activities in the human Hep G2 hepatoma cell line
Biochem. Pharmacol.
Evidence for different ABC-transporters in CaCo-2 cells modulating drug uptake
Pharm. Res.
Genetic linkage of lung cancer-associated MSPI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P450IA1 gene
J. Biochem.
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