Microsomal Cytochrome P450 Enzymes Research Articles

Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity

BackgroundMetabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur.MethodsVarious concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent.ResultsAll the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 – 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested.ConclusionTherefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Identification and kinetics of microsomal and recombinant equine liver cytochrome P450 enzymes responsible for in vitro metabolism of omeprazole

In humans, omeprazole is metabolised by cytochrome P450 (CYP450) CYP2C19 and CYP3A4 with differences in CYP2C19 genotypes leading to variable response to therapy. Despite a wide use of omeprazole in horses with evidence of variable therapeutic efficiency, information regarding enzymatic metabolism is not currently available. This study aims to describe the in vitro kinetics of omeprazole metabolism and determine which enzyme(s) are responsible for omeprazole metabolism in horses. Omeprazole (0–800 uM) was incubated with liver microsomes and a panel of equine recombinant CYP450s (eq-rCYP). Metabolite concentrations were quantified by LC-MS and the kinetics of metabolites’ formation were calculated by non-linear regression analysis. The in vitro liver microsomes formed three metabolites (5-hydroxy-omeprazole, 5-O-desmethyl-omeprazole and omeprazole-sulfone). The 5-O-desmesthyl-omeprazole formation was best fitted to a two enzyme Michaelis-Menten (MM) model with the high affinity site Clint double that of the low affinity site. For 5-hydroxy-omeprazole the best fit was to a 1 enzyme MM model with a Clint higher than for 5-O-desmesthyl-omeprazole (0.12 vs 0.09 pmol/min/pmol P450). The formation of omeprazole-sulfone was negligible. Recombinant CYP3A89 and CYP3A97 produced substantial amounts of 5-hydroxy-omeprazole (1551.72 ng/mL and 1665.33 ng/mL, respectively), while 5-O-desmethyl-omeprazole and omeprazole-sulfone were formed to a much lesser extent by multiple eq-rCYP from the CYP2C and CYP3A family. In vitro metabolism of omeprazole in horses is different to that in humans, with major metabolites produced by the CYP3A family. The current study provides the basis for further investigations of CYP450 single nucleotide polymorphisms that could affect omeprazole metabolism and therapeutic efficacy.

Endothelial deletion of the cytochrome P450 reductase leads to cardiac remodelling.

The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR-/-). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR-/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR-/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.

The dichloroacetamide safener benoxacor is enantioselectively metabolized by monkey liver microsomes and cytosol

The metabolism and toxicity of current-use herbicide safeners remain understudied. We investigated the enantioselective metabolism of the safener benoxacor in Rhesus monkey subcellular fractions. Benoxacor was incubated with liver microsomes and cytosol from female and male monkeys (≤30 min). Benoxacor levels and enantiomeric fractions were determined with gas chromatography. Benoxacor was metabolized by microsomal cytochrome P450 enzymes (CYPs), cytosolic glutathione-S-transferases (GSTs), and microsomal and cytosolic carboxylesterase (CESs). CES-mediated microsomal metabolism followed the order males > females, whereas the CYP-mediated clearance followed the order females > males. CYP-mediated metabolism initially resulted in an enrichment of the second eluting benoxacor enantiomer (E2-benoxacor), whereas the first eluting benoxacor enantiomer (E1-benoxacor) was enriched after 10 or 30 min in female or male microsomal incubations. Benoxacor metabolism by GSTs was enantiospecific, with a total depletion of E1-benoxacor after approximately 20 min. Thus, the enantioselective metabolism of benoxacor by GSTs and CYPs may affect its toxicity.

Molecular Characterization of the Cytochrome P450 Epoxidase (CYP15) in the Swimming Crab Portunus trituberculatus and Its Putative Roles in Methyl Farnesoate Metabolism.

CYP15, which encodes a microsomal cytochrome P450 enzyme, could be involved in juvenile hormone biosynthesis in insects. In this study, a full-length cDNA of CYP15 was cloned from the swimming crab Portunus trituberculatus. This PtCYP15 amino acid sequence contains six conserved domains, which is a typical feature of the cytochrome P450 family. Phylogenetic tree analysis results showed that PtCYP15 clusters in a single branch of crustacean species, suggesting that CYP15 may be more widely present in crustaceans. The PtCYP15 mRNA has a broad pattern of tissue expression in P. trituberculatus, including high levels of expression in the hepatopancreas of both sexes and in the ovary of female crabs. During ovarian development stages, PtCYP15 mRNA is highly expressed in stages I and II and less so in stages III and IV in the hepatopancreas and the ovary of the female crabs. These expression profiles are opposite those of methyl farnesoate in hemolymph, suggesting that PtCYP15 might be involved in methyl farnesoate metabolism. In vitro studies show that only methyl farnesoate upregulated vitellogenin expression in the hepatopancreas, suggesting that methyl farnesoate might be the equivalent of juvenile hormone III in crustaceans. Methyl farnesoate treatment increased levels of PtCYP15 in explants of the hepatopancreas and ovary, while juvenile hormone III treatment reduced levels of PtCYP15 mRNA in ovary explants, suggesting that PtCYP15 might be involved in degrading methyl farnesoate. Furthermore, PtCYP15 mRNA expression levels were inhibited by adding juvenile hormone III to ovary explants. These findings provide foundational information for future research on methyl farnesoate metabolism in crustaceans.

Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis.

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

Metabolism of oral turinabol by the human brain cholesterol 24-hydroxylase CYP46A1

The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17β-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16β-hydroxy oral turinabol by NMR.

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Futile cycling by human microsomal cytochrome P450 enzymes within intact fission yeast cells

Human cytochrome P450 enzymes (CYPs or P450s) are known to be reduced by their electron transfer partners in the absence of substrate and in turn to reduce other acceptor molecules such as molecular oxygen, thereby creating superoxide anions (O2−•). This process is known as futile cycling. Using our previously established fission yeast expression system we have monitored cells expressing each one of the 50 human microsomal CYPs in the absence of substrate for oxidation of dihydroethidium in living cells by flow cytometry. It was found that 38 of these display a statistically significant increase in O2−• production. More specifically, cells expressing some CYPs were found to be intermediate strength O2−• producers, which means that their effect was comparable to that of treatment with 3 mM H2O2. Cells expressing other CYPs had an even stronger effect, with those expressing CYP2B6, CYP5A1, CYP2A13, CYP51A1, or CYP1A2, respectively, being the strongest producers of O2−•.

Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity.

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 μg microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches – formerly indicated to be involved in CPR:CYP interactions – or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Rational evolution of the cofactor-binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes.

NADPH-cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor-linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH. Here, we report a rational evolution approach to enhance the activity of CPR with NADH, in which mutations were introduced into the NADPH-binding flavin adenine dinucleotide (FAD) domain. Multiple CPR mutants that used NADH more effectively than the wild-type CPR in the reduction of the surrogate electron acceptor, cytochrome c were found. However, most were inactive in supporting P450 activity, arguing against the use of cytochrome c as a surrogate electron acceptor. Unexpectedly, several mutants showed significantly improved activity towards CYP2C9 (mutant 1-014) and/or CYP2A6 (mutants 1-014, 1-015, 1-053 and 1-077) using NADPH, even though the mutations were introduced at locations remote from the putative CPR-P450 interaction face. Therefore, mutations at sites in the FAD domain of CPR may be promising future engineering targets to enhance P450-mediated substrate turnover. ENZYMES: NADPH-cytochrome P450 reductase - EC 1.6.2.4; cytochrome P450 - EC 1.14.14.1.

Isoform‐Dependent Effects of Cytochrome P450 Oxidoreductase Polymorphisms on Drug Metabolism by Cytochrome P450 Enzymes in Dogs

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 (CYP) enzymes. We have recently discovered three polymorphisms in the dog POR coding region resulting in one (POR‐H2 and POR‐H4) or two (POR‐H3) amino acid substitutions. In humans, POR mutations can have CYP isoform‐dependent effects on CYP enzyme activity depending on the POR amino acid residue that is substituted.1 Genotyping of 2,040 DNA samples collected from 65 different dog breeds revealed a heterogeneous distribution of POR alleles across breeds. The POR‐H2 allele was found in 19 of 65 breeds and was most common in Rottweilers and Doberman Pinschers (51% and 19% allele frequencies, respectively). The POR‐H3 allele was found in 8 of 65 breeds and was most common in Scottish Deerhounds and Greyhounds (36% and 35% allele frequencies, respectively). The POR‐H4 allele was rare, only being found in 5 of 2,040 dogs. Greyhounds have been shown to metabolize substrates of CYP2B11 (but not other CYPs including CYP2D15) much more slowly compared with mixed‐breed dogs.2 Preliminary studies identified polymorphisms in the CYP2B11 gene that only partially explained reduced CYP2B11 metabolism in Greyhounds. Consequently, we hypothesized that the newly discovered canine POR variants found in Greyhounds would reduce CYP metabolism in a CYP isoform‐dependent manner. The aim of this study was to determine the functional impact of the canine POR variants on drug metabolism by CYP2B11 and CYP2D15 in vitro. In silico molecular structure modelling predicted that POR‐H3 and POR‐H4 but not POR‐H2 would have altered function. The three POR variants and wild‐type were expressed alone or co‐expressed with CYP2B11 or CYP2D15 using an insect cell system. Enzyme kinetic experiments were conducted to determine the impact of the mutations on POR function and CYP‐catalyzed reactions. The POR variants were not significantly different from wild‐type POR in protein expression or function (cytochrome c reduction) when expressed alone. POR variants also did not significantly alter CYP2D15 protein level or catalytic activities or CYP2B11 protein level when co‐expressed. However, POR‐H3 and POR‐H4 (but not POR‐H2) significantly reduced CYP2B11 catalytic efficiency (CLint) by 37% and 69%, respectively, compared to wild‐type POR. This was due to reduced maximal velocities (Vmax) but not substrate affinities (Km). POR‐H3 and POR‐H4 appear to reduce CYP activities in a CYP isoform‐dependent manner. Further investigations are needed to determine POR variant effects on other CYP isoforms and redox partners. The POR‐H3 and POR‐H4 variants are likely to contribute to slower CYP2B11 drug metabolism in Greyhounds and possibly related sighthound breeds (such as Scottish Deerhounds), but results must be confirmed in vivo.Support or Funding InformationAmerican Kennel Club Canine Health Foundation (Grants 2242 and 2529) and the William R. Jones Endowment at Washington State University College of Veterinary Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CYP enzymes, expressed within live human suspension cells, are superior to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P toxicity and cisplatin resistance

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in ‘suspension’ for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3–1.7 μM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.

Microsomal cytochrome P450 enzyme activities in nonalcoholic steatohepatitis livers

Microsomal cytochrome P450 enzyme activities in nonalcoholic steatohepatitis livers

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver.Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

In Vitro Antioxidant Potential and Effect of a Glutathione-Enhancer Dietary Supplement on Selected Rat Liver Cytochrome P450 Enzyme Activity.

Background There is considerable evidence that many people take dietary supplements including those of herbal origin as an alternative therapy to improve their health. One such supplement, with an amalgam of constituents, is CellGevity®. However, the effect of this dietary supplement on drug-metabolizing enzymes is poorly understood, as it has not been studied extensively. Therefore, we investigated the effect of CellGevity dietary supplement on selected rat liver microsomal cytochrome P450 (CYP) enzymes, the most common drug-metabolizing enzymes. We also determined the total antioxidant potential of this dietary supplement in vitro. Methods To determine the antioxidant potential of CellGevity dietary supplement, 2,2-diphenyl-2-picryl-hydrazyl (DPPH), total phenolic, and flavonoid assays were used after initial preparation of a solution form of the supplement (low dose, LD; 4 mg/kg and high dose, HD; 8 mg/kg). Rats received oral administration of these doses of the supplement for 7 days, after which the effect of the supplement on selected liver CYP enzymes was assessed using probe substrates and spectroscopic and high-performance liquid chromatographic methods. Rats which received daily administration of 80 mg/kg of phenobarbitone and distilled water served as positive and negative controls, respectively. Results The IC50 value of the supplement 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. The supplement significantly inhibited rat CYP2B1/2B2 (LDT 92.4%; HDT 100%), CYP3A4 (LDT 81.2%; HDT 71.7%), and CYP2C9 (LDT 21.7%; HDT 28.5%) while it had no significant inhibitory effect on CYPs 1A1/1A2, CYP1A2, and CYP2D6. Conclusion CellGevity dietary supplement possesses moderate antioxidant activity in vitro and has an inhibitory effect on selected rat liver CYP enzymes, suggesting its potential interaction with drugs metabolized by CYP enzymes.

In Vivo Modulation of Rat Liver Microsomal Cytochrome P450 Activity by Antimalarial, Anti-HIV, and Antituberculosis Plant Medicines.

Drug interactions are key reasons for adverse drug reactions and attrition from market. Major infectious diseases causing morbidity/mortality in Ghana are malaria, tuberculosis, and HIV/AIDS. In this study, plant medicines commonly used to treat/manage these diseases in Ghana were investigated for their potential to modulate rat cytochrome P450 enzyme activities. Fluorescence and high-performance liquid chromatography–based assays were used to assess effects of antimalarial plant medicines, Fever (FEV), Mal-TF (MAL), and Kantinka terric (KT); anti-TB medicines, Chestico (CHES), CA + ST Pains + HWNT (TF), and Kantinka herbatic (KHB); and anti-HIV/AIDS medicines, Wabco (WAB), AD + T/AD (LIV) and Kantinka BA (KBA) on rat liver microsomal cytochrome P450 enzyme activities. Effects of medicines on rat biochemical and hematological parameters were also assessed. Generally, the medicines altered microsomal CYP1A1/1A2, CYP2B1/2B2, CYP2C9, and CYP2D6 activities. Only KBA elicited an increase (80%) in CYP1A1/1A2 activity. FEV, MAL, CHES, WAB, and LIV strongly inhibited the enzyme activity. All the medicines significantly inhibited CYP2C9 (24%-80%) activity. CYP2D6 activity increased after treatment with MAL, KBA, LIV, and TF. Also, MAL, WAB, LIV, KHB, and CHES increased CYP2B1/2B2 activity, while KT decrease the activity. Generally, the medicines altered liver function in the rats. Cholesterol levels declined after KBA treatment only. White and red blood cell counts, hemoglobin and hematocrit levels were significantly reduced in KT- and KBA-treated rats. Our results suggest that use of the medicines could have implications for drug interactions and safety, particularly if the medicines are administered over prolonged periods. Further investigations are imperative to establish clinical relevance of these results.

Polymorphisms in cytochrome P450 oxidoreductase and its effect on drug metabolism and efficacy.

Cytochrome P450 oxidoreductase (POR) has played a potential role in the metabolism of drugs and steroids by supplying electrons to microsomal cytochrome P450 (CYP) enzymes. More than 200 different POR mutations and polymorphisms causing more than 130 amino acid changes in the POR protein have been reported since 2004. A503V is a common amino acid sequence variant encoded by POR*28, whereas A287P and R457H are the most common disease-causing mutations in Europeans and Asians, respectively. Polymorphisms in the POR gene can affect POR activity, CYP-mediated drug metabolism activities, and the efficacy of several clinically used drugs. The effects of POR variants on CYP activities are substrate dependent. In this review, recent research on the effects of POR genetic polymorphisms on drug metabolism and therapy has been summarized and discussed, which can contribute to the rational use of drugs in clinic and the development of personalized medicine.