Metabolic Biotransformation Research Articles

Site of metabolism prediction for six biotransformations mediated by cytochromes P450

One goal of metabolomics is to define and monitor the entire metabolite complement of a cell, while it is still far from reach since systematic and rapid approaches for determining the biotransformations of newly discovered metabolites are lacking. For drug development, such metabolic biotransformation of a new chemical entity (NCE) is of more interest because it may profoundly affect its bioavailability, activity and toxicity profile. The use of in silico methods to predict the site of metabolism (SOM) in phase I cytochromes P450-mediated reactions is usually a starting point of metabolic pathway studies, which may also assist in the process of drug/lead optimization. This article reports the Cytochromes P450 (CYP450)-mediated SOM prediction for the six most important metabolic reactions by incorporating the use of machine learning and semi-empirical quantum chemical calculations. Non-local models were developed on the basis of a large dataset comprising 1858 metabolic reactions extracted from 1034 heterogeneous chemicals. For validation, the overall accuracies of all six reaction types are higher than 0.81, four of which exceed 0.90. In further receiver operating characteristic (ROC) analyses, each of the SOM model gave a significant area under curve (AUC) value over 0.86, indicating a good predicting power. An external test was made on a previously published dataset, of which 80% of the experimentally observed SOMs can be correctly identified by applying the full set of our SOM models. The program package SOME_v1.0 (Site Of Metabolism Estimator) developed based on our models is available at http://www.dddc.ac.cn/adme/myzheng/SOME_1_0.tar.gz.

Modeling bioaccumulation using characteristic times

A new formulation of existing mass balance models for bioaccumulation is derived and applied to organisms that respire either water or air. This model employs characteristic time parameters and equations that are mathematically equivalent to those used in existing concentration-rate constant and fugacity models. The equivalence of these traditional formulations and the novel formulation is demonstrated. In all three formulations, the required information includes various physiological and dietary parameters as well as chemical concentrations in food and in the respired medium of water or air. Chemical properties are described by the octanol-water or octanol-air partition coefficient and a metabolic biotransformation half-life. Bioaccumulation, biomagnification, and all uptake and loss rates are expressed using characteristic times that have readily identifiable chemical or biological significance. The ability of the characteristic time formulation to provide an evaluation of the bioenergetic consistency of organism properties is briefly discussed. The model is applied illustratively to a trout as a water-respiring organism and to a wolf as an air-respiring organism, and the results are discussed. It is concluded that the use of characteristic time parameters and equations provides valuable additional insights regarding the relative importance of the various uptake and loss processes and, thus, is complementary to the conventional approaches for modeling bioaccumulation phenomena in a variety of organisms.

A database of fish biotransformation rates for organic chemicals

Biotransformation is a key process that can mitigate the bioaccumulation potential of organic substances and is an important parameter for exposure assessments. A recently published method for estimating whole-body in vivo metabolic biotransformation rate constants (kM) is applied to a database of measured laboratory bioconcentration factors and total elimination rate constants for fish. The method uses a kinetic mass balance model to estimate rates of chemical uptake and elimination when measured values are not reported. More than 5400 measurements for more than 1000 organic chemicals were critically reviewed to compile a database of 1535 kM estimates for 702 organic chemicals. Biotransformation rates range over six orders of magnitude across a diverse domain of chemical classes and structures. Screening-level uncertainty analyses provide guidance for the selection and interpretation of kM values. In general, variation in kM estimates from different routes of exposure (water vs diet) and between fish species is approximately equal to the calculation uncertainty in kM values. Examples are presented of structure-biotransformation relationships. Biotransformation rate estimates in the database are compared with estimates of biodegradation rates from existing quantitative structure-activity relationship models. Modest correlations are found, suggesting some consistency in biotransformation capabilities between fish and microorganisms. Additional analyses to further explore possible quantitative structure-biotransformation relationships for estimating kM from chemical structure are encouraged, and recommendations for improving the database are provided.

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

UDP-glucuronosyltransferases (UGTs), expressed in various tissues including liver and intestine, catalyze phase II metabolic biotransformation. There is little information on species differences between mice and rats in UGT activities, especially in intestine. The purpose of the present study was to clarify the species differences between mice and rats in UGT activities using duodenal and liver microsomes. For estradiol 3-O-glucuronidation in duodenal microsomes, the kinetic data in mice were fit to the Hill equation. However, the Hill coefficient was low in rats (n = 1.1), suggesting that rat estradiol 3-O-glucuronidation followed the Michaelis-Menten equation rather than the Hill equation. For 4-nitrophenol (4-NP) O-glucuronidation, the K(m) values were different between mice and rats. The intrinsic clearance (CL(int)) values for mycophenolic acid (MPA) O- and morphine 3-O-glucuronidation in male mouse duodenum were 3- and 17-fold lower than those in rat, respectively. In male liver, the CL(int) values for 4-NP O-, propofol O-, MPA O-, and morphine 3-O-glucuronidation and the CL(max) value for 4-methylumbelliferone O-glucuronidation in mice were higher than those in rats. The CL(max) value for estradiol 3-O-glucuronidation in mice was lower than that in rats. Also, there were strain differences among C57BL/6J, BALB/c, C3H/HeJ, DBA/2, ddY, and ICR mice in UGT activities in duodenum. We clarified that the species differences in UGT activity evaluated by the CL(int) or CL(max) values in liver and duodenum varied according to the substrate.

Estimating metabolic biotransformation rates in fish from laboratory data

A method is proposed for estimating metabolic biotransformation rate constants for nonionic organic chemicals from measured laboratory bioconcentration and dietary bioaccumulation data in fish. Data have been selected based on a quality review to reduce uncertainty in the measured values. A kinetic mass balance model is used to estimate rates of chemical uptake and elimination. Biotransformation rate constants are essentially calculated as the difference between two quantities, a measured bioconcentration factor or elimination rate constant, and a model-derived bioconcentration factor or elimination rate constant estimated assuming no biotransformation. Model parameterization exploits key empirical data when they are available and assumes default values when study specific data are unavailable. Uncertainty analyses provide screening level assessments for confidence in the biotransformation rate constant estimates. The uncertainty analyses include the range for 95% of the predicted values and 95% confidence intervals for the calculated biotransformation values. Case studies are provided to illustrate the calculation and uncertainty methods. Biotransformation rate constants calculated by the proposed method are compared with other published estimates for 31 chemicals that range in octanol-water partition coefficients from approximately 10(1) to 10(8) and represent over four orders of magnitude in biotransformation potential. The comparison of previously published values with those calculated by the proposed method shows general agreement with 82% of the estimated values falling within a factor of three.

Disposition of Vorinostat, A Novel Histone Deacetylase Inhibitor and Anticancer Agent, in Preclinical Species

The disposition of vorinostat, an anticancer agent, was investigated in rats and dogs. Vorinostat possessed high serum clearance, a short elimination half-life and low oral bioavailability in both species. The renal route played an important role in the elimination of drug-related material and vorinostat was eliminated primarily by metabolic biotransformation.

Estimating Metabolic Biotransformation Rates in Fish from Laboratory Data

Estimating Metabolic Biotransformation Rates in Fish from Laboratory Data

Metamorphosis increases biotransformation of polychlorinated biphenyls: A comparative study of polychlorinated biphenyl metabolism in green frogs (Rana clamitans) and leopard frogs (Rana pipiens) at various life stages

Polychlorinated biphenyl (PCB) elimination rates were used to determine whether green frogs (Rana clamitans) and leopard frogs (Rana pipiens) at three life stages (tadpole, metamorph, and adult) were capable of metabolic biotransformation of PCBs. Polychlorinated biphenyls were grouped according to their structural properties to determine whether congeners with specific structures were eliminated more rapidly than others, which would suggest the occurrence of metabolic biotransformation. For both species and at all life stages studied, PCBs with adjacent unsubstituted meta-para sites (group-2 PCBs) were eliminated significantly faster than PCBs with adjacent unsubstituted ortho-meta sites (group-3 PCBs). Because these groups of PCBs span similar hydrophobicity ranges, it was concluded that green and leopard frogs can selectively metabolize group-2 PCBs. Metabolic activity was more pronounced in the metamorph stage than in any other life stage. In green frog metamorphs, the average elimination rate of the group-2 PCBs was 4.9-fold greater than that of the group-3 PCBs. By contrast, in green frog tadpoles, group-2 PCBs were eliminated 1.9-fold faster than group-3 PCBs, and in green frog adults, group-2 PCBs were eliminated 1.5-fold faster than group-3 PDBs.

Determination of polychlorinated biphenyl and polycyclic aromatic hydrocarbon elimination rates in adult green and leopard frogs

The purpose of the present study was to quantify elimination kinetics of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in adult green frogs (Rana clamitans) and leopard frogs (Rana pipiens). Three experiments were conducted: PCB elimination rate constants were determined for both frog species, and PAH elimination rate constants were determined for leopard frogs only. In green frogs, significant PCB elimination rate constants ranged from 0.013 to 0.04 d(-1) (time for frogs to achieve 90% steady state with water [t90] = 57.8-178.2 d). In leopard frogs, significant PCB elimination rate constants ranged from 0.004 to 0.047 d(-1) (t90 = 48.8-657.9 d). Polycyclic aromatic hydrocarbon elimination in leopard frogs was faster than PCB elimination in either frog species: Significant PAH rate constants ranged from 0.069 to 0.188 d(-1) (t90 = 12.2-33.5 d). In both species, and for both PCBs and PAHs, a significant inverse relationship was found between the chemical elimination rate constant and Kow. These results show that adult anurans have relatively low elimination rates of PCBs but exhibit a small capacity for metabolic biotransformation of PAHs that is comparable to that of invertebrates but lower than that of fish. These findings suggest that adult amphibians have the potential to be used as biomonitors for persistent organic chemicals.

COMPARATIVE METABOLISM OF GERANYL NITRILE AND CITRONELLYL NITRILE IN MOUSE, RAT, AND HUMAN HEPATOCYTES

Geranyl nitrile (GN) and citronellyl nitrile (CN) are fragrance components used in consumer and personal care products. Differences in the clastogenicity of these two terpenes are postulated to result from differential biotransformation, presumably involving the conjugated nitrile moiety. The metabolic clearance and biotransformation of GN and CN were compared in primary hepatocytes from mice, rats, and humans. For determination of intrinsic clearance, GN and CN were incubated with hepatocytes in sealed vials, and the headspace was sampled periodically by solid-phase microextraction and analyzed by gas chromatography/mass spectrometry. For metabolite identification, GN and CN were incubated with hepatocytes from each species for 60 min, and reaction mixtures were extracted and analyzed by mass spectroscopy. Both GN and CN were rapidly metabolized in hepatocytes from all species (T1/2, 0.7-11.6 min). Within a species, intrinsic clearance was similar for both compounds and increased in the order human < rat << mouse. Major common pathways for biotransformation of GN and CN involved 1) epoxidation of the 6-alkenyl moiety followed by conjugation with glutathione, 2) hydroxylation of the terminal methyl group(s) followed by direct conjugation with glucuronic acid in rodents or further oxidation to the corresponding acid in human cells, and 3) hydroxylation of the allylic C5 position. No evidence for either phase I or phase II metabolism of the conjugated nitrile moiety was obtained. Thus, the presumed metabolic basis for differences in genotoxicity remains elusive.

Comparison of supercritical fluid chromatography and liquid chromatography for the separation of urinary metabolites of nobiletin with chiral and non-chiral stationary phases

Nobiletin (NOB), a polymethoxylated flavone found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes, yielding multiple hydroxylated metabolites. However, it has not been possible to date to separate the two demethylated nobiletin metabolites, 3'-demethyl-NOB and 4'-demethyl-NOB (regio-isomers) on reversed-phase liquid chromatography (RPLC). Additionally, both display similar mass spectrometric fragmentation, resulting in difficulties to identify the dominant metabolite. A successful separation method was developed by utilizing supercritical fluid chromatography (SFC) with chiral stationary phase. The separation was also attempted with normal-phase liquid chromatography (NPLC) in both chiral and non-chiral modes. Chromatographic separation for the two nobiletin metabolites was superior by SFC than by LC, especially using chiral stationary phase. By comparing the SFC profile of the synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4'-demethyl-NOB, with the concentration of 28.9 microg/mL.

Metabolic Engineering of Plant Cells for Biotransformation of Hesperedin into Neohesperidin, a Substrate for Production of the Low-Calorie Sweetener and Flavor Enhancer NHDC

Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.

MetaSite: Understanding Metabolism in Human Cytochromes from the Perspective of the Chemist

Identification of metabolic biotransformations can significantly affect the drug discovery process. Since bioavailability, activity, toxicity, distribution, and final elimination all depend on metabolic biotransformations, it would be extremely advantageous if this information could be produced early in the discovery phase. Once obtained, this information can help chemists to judge whether a potential candidate should be eliminated from the pipeline or modified to improve chemical stability or safety of new compounds. The use of in silico methods to predict the site of metabolism in phase I cytochrome-mediated reactions is a starting point in any metabolic pathway prediction. This paper presents a new method, specifically designed for chemists, that provides the cytochrome involved and the site of metabolism for any human cytochrome P450 (CYP) mediated reaction acting on new substrates. The methodology can be applied automatically to all the cytochromes for which 3D structure is known and can be used by chemists to detect positions that should be protected in order to avoid metabolic degradation or to check the suitability of a new scaffold or prodrug. The fully automated procedure is also a valuable new tool in early ADME-Tox assays (absorption, distribution, metabolism, and excretion toxicity assays), where drug safety and metabolic profile patterns must be evaluated as soon, and as early, as possible.

Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation

Ochratoxin A (OTA) is nephrotoxic to all animal species, carcinogenic for rats and mice and probably implicated in human Balkan endemic nephropathy and the associated urothelial tract tumour. Controversial results concerning genotoxicity and biotransformation of OTA have been generated. By 32P post-labelling technique, a dose- and time-dependent DNA adduct formation is observed in vivo and in vitro. Use of several inducers or inhibitors of biotransforming enzymes (including cytochrome P 450, cyclooxygenase, lipoxygenase, glutathione-S-transferase), demonstrated that OTA is biotransformed into genotoxic derivatives damaging for DNA. Authentic C8dG-OTA standards have been synthesized by photo-oxidation. Both of them (C-C8 & O-C8) co-migrate on TLC with two adducts formed by in vitro incubation of OTA in the presence of kidney microsomes, and in vivo in kidney of pig or rodent fed OTA as well as in kidney and bladder tumour of humans exposed to OTA. Several OTA metabolites have been isolated from tissues or cells treated by OTA. The open ring lactone (OP-OTA) and quinone OTA (OTQ) are genotoxic.

Bioenergetics-Based Modeling of Individual PCB Congeners in Nestling Tree Swallows from Two Contaminated Sites on the Upper Hudson River, New York

A bioenergetics-based model was used to simulate the accumulation of total PCBs and 20 PCB congeners by nestling tree swallows at two contaminated sites on the Upper Hudson River, New York. PCB concentrations in birds were calculated as the sum of inherited residues and those acquired through consumption of contaminated insects. Close agreement between simulations and measured residues in 5-, 10-, and 15-day-old nestlings was obtained when PCB concentrations in the diet were set equal to those in food boli taken from adult birds. These simulations were further optimized by fitting the value of a dietary assimilation efficiency constant. Fitted constants for both sites were similar and averaged about 0.7. An evaluation of model performance for individual congeners provided no evidence of metabolic biotransformation. The results of this study are consistent with a companion effort in which principal components analysis was used to compare PCB congener patterns in insects and in tree swallow eggs, nestlings, and adults. Together, these studies establish a quantitative linkage between nestling tree swallows and the insects that they consume and provide strong support for the use of nestling swallows as a biomonitoring species for exposure assessment.

In Vitro Drug Metabolism Using Liver Microsomes

Metabolic biotransformation of a drug can increase the rate of elimination from the body and have a significant effect on efficacy and safety. Drug candidates are screened early in the discovery process for metabolic stability using liver microsomes. Methods for microsome isolation and characterization and the determination of metabolic stability are presented.

Gene expression of detoxifying enzymes in AhR and Nrf2 compound null mutant mouse

The arylhydrocarbon receptor (AhR) regulates the expression of cytochrome P450 (CYP)-1 gene family members which catalyze xenobiotic Phase I metabolism, while Nrf2 exerts the concerted regulation of Phase II enzyme genes. We generated AhR and Nrf2 compound null mutant mice to examine the integrated function of AhR- and Nrf2-regulated enzymes in detoxification. Furthermore, we used this mouse model, by administering three different classes of chemical inducers, to examine how xenobiotic metabolism may be influenced in the absence of signals transduced by AhR or Nrf2. The compound mutant mice responded only weakly to AhR ligand or Phase II inducer, while they displayed a clear response to phenobarbital, an inducer of the CYP2B family through another, unrelated transcription factor. Here, we report an initial characterization of the AhR-Nrf2 double mutant mice, which may serve as a simplified bioassay system to evaluate xenobiotic toxicity and metabolic biotransformation of various drugs and environmental chemicals.

Plasma-Associated Halogenated Phenolic Contaminants in Benthic and Pelagic Fish Species from the Detroit River

Halogenated phenolic contaminants (HPCs), polychlorinated biphenyls (PCBs), and other organochlorine (OC) contaminants (DDTs, chlordanes, HCH, and octachlorostyrene (OCS)) were determined (ng/g, wet wt basis) in the blood plasma of 6 piscivorous/pelagic- and 7 benthic-feeding fish species from the Detroit River. Seven to twelve hydroxylated (OH) PCB congeners with pentachloro- to nonachloro-substitution, pentachlorophenol (PCP) and 4-OH-heptachlorostyrene (4-OH-HpCS), as well as a number of other unidentified HPCs, were detected in all the fish species studied. The concentrations of sigma-OH-PCB (0.57-129.5 ng/ g), 4-OH-HpCS (0.02-0.31 ng/g), and PCP (0.05-3.42 ng/ g) in comparison to other major polychlorinated contaminants (sigma-PCB (10.4-909.0 ng/g), sigma-chlordane (0.72-8.68 ng/g), sigma-DDT (2.10-66.57 ng/g), and sigma-HCH (0.04-0.84 ng/g)) demonstrate the importance of HPCs, especially OH-PCBs, as plasma-associated contaminants. OH-PCBs are most likely metabolites of PCBs, and 4-OH-HpCS is a likely metabolite of OCS. Our findings indicate that metabolic biotransformation is a factor in the bioaccumulation, pharmacokinetics, and fate of OCS and highly chlorinated PCB congeners that are generally more recalcitrant in fish. Target tissue exposure to circulating OH-PCBs was variable, and thus potential OH-PCB-mediated toxicological activity and effects (e.g., endocrine-related) and health risks are different among the fish species.

Non-steroidal anti-inflammatory drugs in chemoprevention of breast and prostate cancer

Despite convincing evidence from animal experiments, epidemiological studies linking the use of non-steroidal anti-inflammatory drugs (NSAIDs) with lower risk of breast and prostate cancer have been equivocal. One explanation for the inconsistencies among epidemiological studies may relate to individual differences in NSAID metabolism due to genetic polymorphisms in enzymes such as N -acetyltransferases and cytochrome P4502C9, which are known to be involved in the metabolic biotransformation of NSAIDs. The exclusion of these molecular biomarkers of individual susceptibility may have contributed to the inconsistent findings on the effects of NSAIDs in breast and prostate cancer.

In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues

Malathion [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorodithioate] is a commonly used organophosphorus insecticide reported to be genotoxic both in vivo and in vitro, but the reports are conflicting. In order to elucidate the genotoxic potency of the main compounds present in commercial preparations of malathion, the DNA-damaging effect of this insecticide, its major metabolite malaoxon [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorothiolate] and its isomer isomalathion [S-(1,2-dicarboethoxyethyl)O,S-dimethyl phosphorodithioate], all at purity of at least 99.8%, was investigated by use of the alkaline single cell gel electrophoresis (comet assay). Freshly isolated human peripheral blood lymphocytes were incubated with 25, 75 and 200 μM of the chemicals for 1 h at 37°C. The concentrations used are comparable to those found in blood following various non-lethal human exposures to pesticides. Malathion did not cause any significant changes in the comet length of the lymphocytes, throughout the range of concentrations tested. Malaoxon and isomalathion introduced damage to DNA in a dose-dependent manner. The effect induced by malaoxon was more pronounced than that caused by isomalathion. Treated cells were able to recover within a 60-min incubation in insecticide-free medium at 37°C except the lymphocytes exposed to malaoxon at 200 μM, which did not show measurable DNA repair. The latter result suggests a considerable cytotoxic effect (cell death) of malaoxon at the highest concentration used. The reported genotoxicity of malathion might, therefore, be a consequence of its metabolic biotransformation to malaoxon or the presence of malaoxon and/or isomalathion as well as other unspecified impurities in commercial formulations of malathion. In this regard, the results of our study clearly indicate that malathion used as commercial product, i.e., containing malaoxon and isomalathion, can be considered as a genotoxic substance in vitro. This means that it may also produce DNA disturbances in vivo, such as DNA breakage at sites of oncogenes or tumor suppressor genes, thus playing a role in the induction of malignancies in individuals exposed to this agent. Therefore, malathion can be regarded as a potential mutagen/carcinogen and requires further investigation.