Human CYP2B6 Research Articles

The impact of the clinical CYP11B2 mutation V386A strongly depends on the enzyme's genetic background.

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

Emerging roles of human CYP1B1 in cancer growth and metastasis

Emerging roles of human CYP1B1 in cancer growth and metastasis

Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes.

MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4.

PS 04-27 EFFECT OF HIGH SALT DIET ON THE EPIGENETICS OF ALDOSTERONE SYNTHASE GENE, CYP11B2

Objective: We found the hypomethylation status of CYP11B2 gene in the aldosterone-producing adenomas compared with normal adrenal glands or non-functioning adrenal adenomas and reported that the methylation of promoter region of this gene influenced the mRNA expression in the adrenal cells. Angiotensin II or potassium influenced the methylation status of CYP11B2 gene. In order to clarify the effect of salt intake on the methylation status of CYP11B2 gene, rats were fed with high or low salt diet and the methylation status of CYP11B2 gene in the adrenal gland was examined. Design and Method: Five Wister rats were fed with high, normal and low salt diet for 8 weeks. Plasma and urinary aldosterone, PRA were measured by RIA. The gene expression of CYP11B2 was measured by real time quantitative PCR in the adrenal glands. Isolated DNAs from rat adrenal glands were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions. Results: Plasma and urinary aldosterone and PRA were significantly decreased by high salt diet compared with normal or low salt diet (p < 0.05). High salt diet significantly decreased mRNA levels of CYP11B2 gene and increased metylation ratio of this gene (p < 0.05). Conclusions: High salt diet may influence the methylation ratio of CYP11B2 gene and regulate aldosterone biosynthesis.

Activation of uPA-uPAR signaling pathway by human CYP1B1 promotes cancer progression and metastasis in human breast cancer cells

Flavonoids are phenolic compounds isolated from plants, and several of them like genistein and quercetin, have been documented to be effective in preventing cancer. Baicalein, a flavonoid extracted from the root of Scutellaria species, is widely used as a health supplement and herbal medicine in Asian countries. In this study, the chemopreventive effect of baicalein on 7,12-dimethylbenz〚a〛anthracene (DMBA)-induced DNA damage was evaluated in an established cell culture model. In a preliminary screening, baicalein was identified to be a strong inhibitor to EROD activities induced by DMBA in MCF-7 cells. Subsequent enzyme kinetic analysis revealed that baicalein was a competitive inhibitor to EROD, and CYP1A1 and CYP1B1 gene expressions were also determined. Baicalein could reduce the CYP1A1/1B1 mRNA expressions induced by DMBA, and the mRNA abundance of CYP1A1 appeared to be more responsive than that of CYP1B1. A XRE–luciferase gene reporter assay indicated that AhR transactivation was suppressed. Since CYP1A1/1B1 were responsible for the biotransformation of polycyclic aromatic hydrocarbons, baicalein also demonstrated its ability to reduce DMBA–DNA adduct formation in MCF-7 cells. This study suggested that the natural occurring baicalein could be an agent preventing carcinogen–DNA adduct formation.

In Vitro Regio- and Stereoselective Oxidation of β-Ionone by Human Liver Microsomes.

The metabolism of the norisoprenoid β-ionone was investigated in vitro using human liver microsomes and 11 different recombinant cytochrome P450 enzymes expressed in Trichoplusia ni cells. β-Ionone was found to be oxidized via 4S-hydroxylation by CYP2B6 in human liver microsomes. CYP1A2 also regioselectively catalyzed the hydroxylation of β-ionone to yield 4-hydroxylation; this conversion was not stereoselective. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for β-ionone 4-hydroxylation. Kinetic analysis showed that Km and Vmax for oxidation of β-ionone by CYP1A2 and CYP2B6 was 107.9 ± 36.0 µM and 3200.3 ± 323.0 nmol/min/nmol P450 and 5.6 ± 1.2 µM and 572.8 ± 29.8 nmol/min/nmol P450, respectively. The reaction rates observed using human liver microsomes and recombinant CYP2B6 were very high compared with those of other CYP2B6 substrates reported thus far. These results suggest that β-ionone, a norisoprenoid present in nature, is one of the effective substrates for CYP2B enzymes in human liver microsomes. To the best of our knowledge, this is the first time that 4-hydroxy β-ionone has been described as a human metabolite of β-ionone.

Roles of Residues F206 and V367 in Human CYP2B6: Effects of Mutations on Androgen Hydroxylation, Mechanism-Based Inactivation, and Reversible Inhibition.

The crystal structures of human CYP2B6 indicate that Phe206 and Val367 are in close proximity to the substrate binding site and suggest that both residues may play important roles in substrate metabolism and inhibitor binding. To test this hypothesis, we investigated the effects of mutating these residues to Ala on the regiospecificity of CYP2B6 for the metabolism of testosterone and androstenedione. For testosterone metabolism, 16β-OH-testosterone formation by the F206A mutant was <5% of the wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-OH-testosterone formation with a 50% decrease in 16β-OH-testosterone formation compared with the WT. Significant alterations in the regiospecificity for androstenedione metabolism were also observed. To investigate the roles of these two residues in the metabolic activation of mechanism-based inactivators, tert-butylphenylacetylene (BPA) and bergamottin (BG) were used to test the susceptibility to inactivation. Although the rates of inactivation of both mutants by BG were not significantly decreased compared with the WT, the efficiency of inactivation by BPA of both mutants was more than an order of magnitude lower. Our results demonstrate that Phe206 plays a crucial role in determining the specificity of CYP2B6 for the 16β-hydroxylation of testosterone and androstenedione and that it also plays an important role in BG binding and mechanism-based inactivation by BPA. In addition, Val367 dramatically enhances the catalytic activity of CYP2B6 toward androstenedione and plays an important role in mechanism-based inactivation by BPA. The results presented here show the important roles of Phe206 and Val367 in interactions of CYP2B6 with substrates and inactivators/inhibitors and are consistent with the crystal structures.

Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

From the Cover: Structural Determinants of the Position of 2,3',4,4',5-Pentachlorobiphenyl (CB118) Hydroxylation by Mammalian Cytochrome P450 Monooxygenases.

Polychlorinated biphenyls (PCBs) accumulate in mammals via the food chain because of their characteristics such as slow degradation and high hydrophobicity. One of the 209 PCB congeners, 2,3',4,4',5-pentachlorobiphenyl (CB118), is abundantly found in the environment and in mammals. Understanding the metabolic fate of CB118 can provide important information toward evaluating its toxicity. In vitro studies on the metabolism of CB118 by cytochrome P450 enzymes (P450 or CYP) revealed that human CYP2B6 and rat CYP2B1 primarily metabolized it to 3-hydroxy (OH)-CB118, whereas rat CYP1A1 metabolized CB118 to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). Docking models of CYP2Bs with CB118 revealed a short distance between the 3-position of CB118 and the heme iron caused by polarization of the substrate-binding cavity, and maintenance of this pose through interaction with the peripheral amino acids determines the activity and position of hydroxylation. 4-Hydroxylation by rat CYP1A1 occurs owing to the longitudinal shape of the substrate-binding cavity toward the heme of CYP1A1. The metabolites 3-OH-CB118 and 4-OH-CB107 decreased potential for activating the aryl hydrocarbon receptor compared with that of CB118, thereby leading to a decrease in dioxin-like toxicity; however, the neurodevelopmental toxicity of 4-OH-CB107 has been previously reported. The results suggest that these 3 P450 isoforms play an important role in determining the extent of CB118 toxicity. This study will contribute to understanding of the metabolic fates and toxicity of CB118 in vivo.

Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation

Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed.

Dibenzo[def,p]chrysene transplacental carcinogenesis in wild-type, Cyp1b1 knockout, and CYP1B1 humanized mice.

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (P = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild-type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755-762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60-69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis. © 2016 Wiley Periodicals, Inc.

Characterization of feline cytochrome P450 2B6

1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung.2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human.3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver.4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6.5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings.

Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.

Regulation of drug-metabolizing enzymes and efflux transporters by Astragali radix decoction and its main bioactive compounds: Implication for clinical drug–drug interactions

Ethnopharmacological relevanceAstragali radix (“Huang Qi” in Chinese, HQ) is a well-known traditional Chinese herbal medicine that possesses various biological functions. Astragaloside IV (AS-IV), calycosin (CS), and formononetin (FMNT) are the three main bioactive compounds of HQ that are responsible for its pharmacological activities and therapeutic efficacy. Aim of the studyThis study aims to investigate the effects of HQ, AS-IV, CS, and FMNT on major human drug-metabolizing enzymes (DMEs), including CYP3A4, CYP2B6, CYP2E1, UGT1A, UGT1A6, SULT1A1, and SULT1A3, as well as efflux transporters (ETs), including P-gp, MRP2, BCRP, MRP1, and MRP3, by using HepG2 cell line. Results would provide beneficial information for the proper clinical application of HQ. Materials and methodsHepG2 cells were treated with HQ, AS-IV, CS, and FMNT for 96h. Cell viability was examined by MTT assay. The protein and mRNA levels of DMEs and ETs were measured using Western blot and real-time PCR, respectively. ResultsCompared with the control group, HQ considerably increased the expression levels of CYP3A4, CYP2B6, CYP2E1, UGT1A, P-gp, MRP2, BCRP, and MRP3 in a dose-dependent manner. Inversely, HQ significantly decreased the protein levels of UGT1A6, SULT1A1, and MRP1. Exposure to AS-IV induced the protein levels of UGT1A, P-gp, MRP1, and MRP3, but produced inhibitory effects on CYP3A4, CYP2B6, and BCRP. The expression levels of CYP3A4, UGT1A, SULT1A1, P-gp, MRP2, and MRP3 were remarkably increased in the CS-treated cells, whereas the protein levels of SULT1A3 and BCRP were decreased. FMNT treatment induced the protein levels towards CYP3A4, CYP2B6, UGT1A, P-gp, MRP1, MRP2, and MRP3, but inhibited the expression of CYP2E1, SULT1A1, and SULT1A3. ConclusionsHQ and its main bioactive compounds, including AS-IV, CS, and FMNT significantly regulated the expression of the major DMEs and ETs. HQ produced stronger regulations (induction or inhibition) on DMEs and ETs than AS-IV, CS, or FMNT alone. The results indicate that potential drug–drug interactions might exist when the tested drugs, specifically HQ, are co-administered with other substrate drugs that are metabolized or transported via the studied DMEs or ETs. This study provides beneficial information for appropriate use of HQ for clinical therapy.

Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes.

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17β-hydroxy-17α-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.

Establishing Modified Human CYP4B1 As a Novel Suicide Gene System for Adoptive T-Cell Therapies

Retargeting autologous T-cells to recognize tumor-associated antigens by genetic modifications with viral vectors that express either enhanced T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has shown successful activity against B-cells malignancies. Equipping the transduced T-cell with a suicide gene can prevent potential safety problems as on-target off-target toxicity, off-target reactivity and cytokine-release syndrome (Casucci et al., 2015). The concept of a suicide gene has already been utilized to prevent a Graft-versus-host-disease (GvHD) following donor lymphocyte infusion (DLI). The first suicide gene system used in clinical phase I/II studies was the human Herpes simplex virus type 1 thymidine kinase with its prodrug ganciclovir that was used to control Graft-versus-host-disease following donor lymphocyte infusion (DLI) for leukemia relapse after allogeneic stem cell transplantation. More recent examples for suicide gene systems are icasp9, an inducible apoptosis switch with a synthetic dimerizer protein as prodrug, and CD20 with rituximab as a prodrug. We recently published a novel potential human suicide gene system by rendering the inactive P450 cytochrome CYP4B1 with 13 amino acid changes into the highly active CYP4B1P+12 in combination with 4-ipomeanol (4-IPO) (Wiek et al., 2015). 4-IPO is furan produced by sweet potatoes infected with the common mold Fusarium javanicum and has been identified as the major lung toxin causing lung edema in livestock (Wilson et al., 1970). A second natural occurring substance causing lung edema in horses is the major component of the Perilla frutenscens fruit oil called perilla ketone (PK) (Wilson et al., 1977). Importantly, the only difference between PK and 4-IPO is a methyl group at C5 in PK, where 4-IPO has a hydroxyl group. Therefore in this work, we tested the hypothesis whether PK could function as a potential new prodrug for the CYP4B1P+12 suicide gene.Initially we compared the cytotoxicity caused by PK and 4-IPO in CYP4B1P+12 transduced primary human T-cells from healthy donors. For these experiments, we used third generation lentiviral vectors with T2A sites to express the suicide gene in combination with a truncated version of the low affinity nerve growth factor receptor (DNGFR) as a selection marker for Miltenyi MACS microbeads. Through the selection process, we were able to enrich the suicide gene expressing T-cell population from 61±6.0% to ≥95±1.8%.After selection, we challenged the CYP4B1P+12 expressing T-cells with increasing concentrations (2.9-90 µM) of both substrates and analyzed the survival of the cells. After 24h, 65.8±5.26% of CYP4B1P+12 positive T-cells incubated with 2.9 µM 4-IPO were still alive, while incubation with 90 µM lead to only 17.5±1.45% living T-cells. In contrast, incubation with 2.9 µM PK resulted in only 16.02±1.08% living cells, while 90 µM PK induced cell death in 94.2±0.79% of cells. Next, we demonstrated that a lower concentration, 0.9 µM, already led to only 13.2±0.46% alive cells after 24h. Monitoring apoptosis in the transduced T-cells by flow cytometry demonstrated that incubation with 90 µM 4-IPO for 4h decreased the rate of transduced T-cells to only 87.4±2.89%, while 10h and 24h incubation resulted in 20.4±4.9% and 5.8±0.93% living annexinV-negative cells. In contrast, 2h incubation with only 9 µM PK led to 88.7±2.07%, 4h to 60.2±5.47% and 10h to only 5.5±1.85% living annexinV-negative T-cells. Incubation of the T-cells with 90 µM did not change these numbers. Hence, we were able show that PK is not only more potent as 4-IPO, it also proves to have a faster kinetic in inducing apoptosis.After these promising in vitro results of using PK as a novel more potent prodrug for the CYP4B1P+12 human suicide gene, we performed dose escalation studies of PK in mice with targeted disruption of the Cyp4b1 gene. Using up to 25 mg PK/kg mouse daily up for 3d did not induce any clinical symptoms in the Cyp4b1-/- animals, while Cyp4b1+/+ animals needed to be sacrificed 4h and 7h after a single i.p. injection of 10 mg/kg or 5 mg/kg PK, respectively, due to severe respiratory distress. Results of in vivo studies analyzing the elimination of CYP4B1P+12 positive syngenic T-cells in Cyp4b1-/- mice after injection with either 4-IPO or PK will be finalized and presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Cyp2b1 gene expression in the rat liver and brain by multiplex PCR

The CYP2B6 enzyme is involved in the metabolism of environmental toxins and endogenous compounds. The expression and activity of the cyp2b6 gene is important for the brain’s ability to detoxify harmful compounds. In rats, the Cyp2b1 gene is homologous to the human Cyp2b6 gene and has an isoform, Cyp2b2, with nearly the same sequence. Although the distinction between them is fundamental, most studies report global changes in CYP2B enzymes due to the complexity of the techniques to detect both genes separately. We instrumented a semi-quantitative technique combining multiplex end point RT-PCR with restriction enzyme assays, allowing us to establish a relatively simple, reproducible and affordable method to distinguish Cyp2b1 gene expression from Cyp2b2. Using this technique, we showed differential Cyp2b1 expression after phenobarbital and ethanol induction in the brain and liver and detected increased Cyp2b1 expression after ethanol treatment in the brain striatum that had not been detected before.

Metabolism of the14C-labeled herbicide clodinafop-propargyl in plant cell cultures of wheat and tobacco

ABSTRACTThe metabolism of 14C-clodinafop-propargyl (CfP) was examined in cell cultures of wheat (Triticum aestivum L. cv. ‘Heines Koga II’) and tobacco (Nicotiana tabacum L.). Besides the non-transgenic tobacco culture, cultures transformed separately with cDNA of human cytochrome P450-monooxygenases (P450s) CYP1A1, CYP1A2, CYP3A4, CYP2B6 and CYP2C19 were examined. Experiments with wheat were executed in the presence and absence of safener cloquintocet-mexyl (CqM). After 48 h of incubation, only about 10% of applied 14C was found in media (both tobacco and wheat). Non-extractable residues of 14C-CfP in wheat cells were 16.54% (without CqM) and 30.87% (with CqM). In all tobacco cultures, 82.41–92.46% of applied radioactivity was recovered in cell extracts. In contrast to wheat, non-extractable residues amounted only to 1.50–2.82%. As determined by radio-thin layer chromatography (TLC) and -high-performance liquid chromatography (HPLC), the parent CfP was not found in the cell extracts of wheat; in tobacco cell extracts, only traces of CfP were detected. After a hydrolysis of assumed carbohydrate conjugates of CfP derived polar 14C-labeled compounds, TLC and HPLC analysis showed that in wheat, a more complex pattern of metabolites of CfP were observed as compared to all tobacco cultures. In hydrolysates resulting from wheat, the identity of three primary products was confirmed by means of GC-EI-MS: free acid clodinafop (Cf), hydroxy-Cf hydroxylated at the pyridinyl moiety, and 4-(5-chloro-3-fluoropyridin-2-yloxy)phenol. In hydrolysates derived from all tobacco cultures, main metabolite was Cf besides only traces of further unidentified products. Differences among the different tobacco cultures (non-transgenic, transgenic) did not emerge. According to kinetics of disappearance of primary metabolite Cf as well as formation of polar soluble products and non-extractable residues, metabolization of CfP proceeded at a noticeably higher rate in wheat cells treated with safener CqM than in cells without CqM treatment. Thus, these results indicated a stimulation of CfP's metabolism by CqM, although metabolic profiles observed in CqM treated and non-treated cells (after hydrolysis) were qualitatively similar. The findings obtained from all tobacco cultures suggested that with the exception of ester cleavage to Cf, CfP cannot be metabolized by tobacco itself or by the human P450s examined.

Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution.

CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5–exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.

Abstract P211: Normo-aldosteronemic Aldosterone-producing Adenoma: Immunochemical Characterization and Diagnostic Implications

Background: Primary Aldosteronism (PA), the most common form of endocrine hypertension, is usually identified by a high aldosterone-renin ratio (ARR) alongside elevated plasma aldosterone concentration (PAC) values. Normo-aldosteronemic PA is also being diagnosed when only the high ARR was driven by low renin because PAC was normal. However, whether this entity truly exists remains contentious since most such cases did not undergo surgical confirmation and even when adrenalectomy was performed no demonstration of an aldosterone-producing adenoma (APA) could be obtained at immunohistochemistry. Case Description: In 2003 a young lady presented with severe hypertension, low plasma renin activity (PRA), but consistently normal PAC values. She undertook a magnetic resonance that revealed a small adenoma on the right side, but adrenal vein sampling (AVS) showed left lateralization of aldosterone secretion. Therefore, she underwent left laparoscopic adrenal surgery, which determined complete normalization of blood pressure and PRA long term follow up (12 years). In 2014 the development of novel monoclonal antibodies for the human aldosterone synthase CYP11B2 and 11β-hydroxylase CYP11B1, allowed us to immunochemically characterize the resected adrenal gland. Double immunostaining for CYP11B2 and CYP11B1 showed a small CYP11B2-positive adenoma, thus unequivocally proving the presence of an APA. Conclusions: This case provides compelling evidence for the existence of normo-aldosteronemic APA and suggests that many cases that we dismiss as “low renin-essential hypertension” might instead have an undetected surgically curable APA.