In vitro human phase I metabolism of xenobiotics I: pesticides and related compounds used in agriculture and public health,May 2003

This is the first revision of a database covering human phase I enzymes and their isoforms that metabolize pesticides and related compounds. The original version included enzymes that metabolize chloroacetamide and triazine herbicides, and organophosphorus insecticides. This revision also includes carbamate, nicotinoid, and pyrethroid insecticides and insect repellents.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol

Carbofuran is a carbamate pesticide used in agricultural practice throughout the world. Its effect as a pesticide is due to its ability to inhibit acetylcholinesterase activity. Though carbofuran has a long history of use, there is little information available with respect to its metabolic fate and disposition in mammals. The present study was designed to investigate the comparative in vitro metabolism of carbofuran from human, rat, and mouse liver microsomes (HLM, RLM, MLM, respectively), and characterize the specific enzymes involved in such metabolism, with particular reference to human metabolism. Carbofuran is metabolized by cytochrome P450 (CYP) leading to the production of one major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in the oxidation to 3-hydroxycarbofuran is significantly less in HLM (K_m = 1.950 mM) than in RLM (K_m = 0.210 mM), or MLM (K_m = 0.550 mM). Intrinsic clearance rate calculations indicate that HLM are 14-fold less efficient in the metabolism of carbofuran to 3-hydroxycarbofuran than RLM or MLM. A screen of 15 major human CYP isoforms for metabolic ability with respect to carbofuran metabolism demonstrated that CYP3A4 is the major isoform responsible for carbofuran oxidation in humans. CYP1A2 and 2C19 are much less active while other human CYP isoforms have minimal or no activity toward carbofuran. In contrast with the human isoforms, members of the CYP2C family in rats are likely to have a primary role in carbofuran metabolism. Normalization of HLM data with the average levels of each CYP in native HLM, indicates that carbofuran metabolism is primarily mediated by CYP3A4 (percent total normalized rate (% TNR) = 77.5), although CYP1A2 and 2C19 play ancillary roles (% TNR = 9.0 and 6.0, respectively). This is substantiated by the fact that ketoconazole, a specific inhibitor of CYP3A4, is an excellent inhibitor of 3-hydroxycarbofuran formation in HLM (IC₅₀: 0.31 μM). Chlorpyrifos, an irreversible non-competitive inhibitor of CYP3A4, inhibits the formation of 3-hydroxycarbofuran in HLM (IC₅₀: 39 μM). The use of phenotyped HLM demonstrated that individuals with high levels of CYP3A4 have the greatest potential to metabolize carbofuran to its major metabolite. The variation in carbofuran metabolism among 17 single-donor HLM samples is over 5-fold and the best correlation between CYP isoform activity and carbofuran metabolism was observed with CYP3A4 (r² = 0.96). The interaction of carbofuran and the endogenous CYP3A4 substrates, testosterone and estradiol, were also investigated. Testosterone metabolism was activated by carbofuran in HLM and CYP3A4, however, less activation was observed for carbofuran metabolism by testosterone in HLM and CYP3A4. No interactions between carbofuran and estradiol metabolism were observed.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Furthermore, a mode of binding interaction for the inhibitor, alpha-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes.     

In vitro metabolism and inductive or inhibitive effect of DL111 on rat cytochrome P4501A enzyme

In vitro metabolism and the inductive or inhibitive effect of DL111, a non-hormonal early pregnancy-terminating agent, toward cytochrome P450 (CYP) enzymes in rat liver microsomes were studied. In vitro metabolism of DL111 was performed in different rat liver microsomes (pretreated with phenobarbital (PB), dexamethasone (Dex), beta-naphthoflavone (BNF), DL111, respectively) and the catalytic abilities of these microsomes for DL111 were compared with control group. DL111 was well metabolized in microsomes pretreated with beta-naphthoflavone and itself. The K(m) and V(max) was 41.76 +/- 3.26 microM and 15.34 +/- 1.03 nM min(-1) mg(-1) protein for beta-naphthoflavone group, 48.17 +/- 6.06 microM and 17.54 +/- 1.79 nM min(-1)mg(-1) protein for DL111 group, 77.81 +/- 4.73 microM and 3.087 +/- 0.202 nM min(-1)mg(-1) protein for control group, respectively. The rats were pretreated intraperitoneally with the same daily dose of DL111 for different days. The DL111-pretreated microsomal enzymatic activities were evaluated by measuring the metabolic abilities for specific substrates of various enzymes. The results showed that DL111 had the same inductive function as beta-naphthoflavone (the specific inducer of CYP1A) toward rat liver microsomes. The inhibitive effect of DL111 on CYP1A was investigated by coincubating DL111 with the specific substrates of CYP1A-ethoxyresorufin or phenacetin in the microsome induced by beta-naphthoflavone, and the inhibitive level was compared with fluvoxamine (Flu), the specific inhibitor of CYP1A. DL111 inhibited significantly the metabolism of phenacetin and ethoxyresorufin with the inhibition constant (K(i)) 6.836 +/- 0.10 and 1.222 +/- 0.230 microM, respectively and its inhibition potential on CYP1A was higher than fluvoxamine.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

The metabolism of nonane, a JP-8 jet fuel component, by human liver microsomes, P450 isoforms and alcohol dehydrogenase and inhibition of human P450 isoforms by JP-8

Nonane, a component of jet-propulsion fuel 8 (JP-8), is metabolized to 2-nonanol and 2-nonanone by pooled human liver microsomes (pHLM). Cytochrome P450 (CYP) isoforms 1A2, 2B6 and 2E1 metabolize nonane to 2-nonanol, whereas alcohol dehydrogenase, CYPs 2B6 and 2E1 metabolize 2-nonanol to 2-nonanone. Nonane and 2-nonanol showed no significant effect on the metabolism of testosterone, estradiol or N,N-diethyl-m-toluamide (DEET), but did inhibit carbaryl metabolism. JP-8 showed modest inhibition of testosterone, estradiol and carbaryl metabolism, but had a more significant effect on the metabolism of DEET. JP-8 was shown to inhibit CYPs 1A2 and 2B6 mediated metabolism of DEET, suggesting that at least some of the components of JP-8 might be metabolized by CYPs 1A2and/or 2B6.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).     

Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes

The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O-sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O-Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC-UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS.     

Xenobiotic metabolizing enzymes in the central nervous system: Contribution of cytochrome P450 enzymes in normal and pathological human brain

The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer's and Parkinson's diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases.     

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography,docking, molecular dynamics,and free energy calculations

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug‐like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti‐inflammatory drugs (NSAIDs). Interestingly, single active‐site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X‐ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free‐energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free‐energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active‐site mutations. Our findings confirm that use of MD and binding free‐energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate. Proteins 2016; 84:383–396. © 2016 Wiley Periodicals, Inc.     

Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2

Primary cultures of human hepatocytes and hepatoma cell line HepG2 are frequently used to evaluate the hepatic disposition of drugs and other xenobiotics. To check the variability of the expression of drug-metabolizing enzymes in these in vitro models, expression of genes coding for several cytochrome P450 isoforms and phase II enzymes was quantified during culture time by real-time RT-PCR. Gene expression was determined daily for primary hepatocytes maintained in a sandwich culture over 1 week and for HepG2, during the first 10 passages. In primary hepatocytes characteristic expression trends were observed which could be abstracted into three major classes of time curves. Genes of the first and the second class had an expression maximum around day 6 and day 4 in culture, respectively. The third class of genes had two expression peaks: at day 1 and 5 in culture. Surprisingly, also the cell line HepG2 showed significant expression changes during passages. For example, gene expression of cytochrome 1A1 varied 8-fold, that of cytochrome 2B6 30-fold, and that of NADP-quinone reductase 1 more than 200-fold within the first 10 passages. In conclusion, neither primary hepatocytes nor HepG2 cell line display a model for constant expression of drug-metabolizing enzymes.