Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy-N,N-dimethyltryptamine metabolism and pharmacokinetics

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency (V_max/K_m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities (R² = 0.98; P < 0.0001) and CYP2D6 contents (R² = 0.77; P = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg-CYP2D6) mouse models showed that Tg-CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg-CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.     

Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo

Ritonavir, an HIV protease inhibitor, is successfully used for the prevention and treatment of HIV infections. Ritonavir pharmacokinetics are complicated by inhibition, induction and pharmacogenetics of cytochrome P450 (CYP) enzymes mediating its clearance. This investigation revealed that CYP2J2, along with CYP3A4/5 and CYP2D6, efficiently metabolizes ritonavir, and to a CYP2J2-specific (minor) metabolite. Chemical inhibition of ritonavir metabolism, clearance, K_I/k_inact and abundance of CYP2J2 in liver microsomes were evaluated and then applied to an in vitro–in vivo static scaling model to estimate the contribution of each isozyme, as a function of CYP abundance, activity, and genotype. Disposition of the CYP2J2-specific metabolite was also evaluated in vivo. In plasma, metabolite abundance was well above previously reported levels with circulating concentrations measured at 2 μM for the main hydroxylisopropyl metabolite. Ritonavir and metabolite plasma profiles were simulated using Simcyp^®. A modest (2–6%) contribution of CYP2J2 to ritonavir clearance is predicted which increases to more than 20% in subjects carrying CYP2D6 poor metabolizer polymorphisms and CYP3A4 irreversible inhibition. These results indicate that minor drug metabolizing enzymes could become quantitatively important in RTV clearance if main metabolic pathways are impeded.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Gentamicin caused renal injury deeply related to methylglyoxal and N-(carboxyethyl)lysine (CEL)

In this study, we investigated the role of carbonyl stress in gentamicin (GM)-induced renal injury in rats. Carbonyl stress is represented by methylglyoxal (MGO) and its downstream advanced glycation end products, such as N^?-(carboxyethyl)lysine (CEL). GM (150 mg /kg/day, i.p.) administration for 6 days significantly increased blood urea nitrogen (BUN) levels from 24.06 ± 0.55 to 85.04 ± 21.31 mg/dL and decreased creatinine clearance rate (Ccr) from 10.68 ± 0.76 to 2.53 ± 1.11 ml/min/kg B.W.; biopsy showed tubular injury. The kidney levels of MGO and CEL increased significantly from 9.56 ± 1.94 to 79.13 ± 17.96 μg/g of protein and from 0.03 ± 0.00 to 0.06 ± 0.00 μmol/μg of protein, respectively. Therefore, MGO and CEL appeared to be associated with GM-induced renal damage. Co-administration of metformin (50 or 100 mg/kg/day) and GM for 13 days effectively reversed GM-induced renal damage. The kidney levels of MGO and CEL decreased significantly from 24.95 ± 7.74 to 22.98 ± 17.74 μg/g of protein and from 0.04 ± 0.01 to 0.03 ± 0.01 μmol/μg of protein (both vs. the GM group), respectively. The identification of this new pathway may help prevent GM-induced nephrotoxicity.     

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 μg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 μg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 μg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24 h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6 h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.     

Inhibition of cytochrome P450 by ethambutol in human liver microsomes

Although cytochrome P450 inhibition is the major drug–drug interaction (DDI) mechanism in clinical pharmacotherapy, DDI of a number of well-established drugs have not been investigated. Rifampicin, isoniazid, pyrazinamide and ethambutol combination therapy inhibits clearance of theophylline in patients with tuberculosis. We determined the inhibitory effects of ethambutol on the activities of nine CYP isoforms including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in pooled human liver microsomes (HLM). As measured by liquid chromatography–electrospray ionization tandem mass spectrometry, ethambutol exhibited strong inhibitory potential against CYP1A2 and CYP2E1, moderate against CYP2C19 and CYP2D6 and weak against CYP2A6, CYP2C9 and CYP3A4, based on the IC₅₀ values. The K_i value of ethambutol for CYP1A2 was 1.4 μM and for CYP2E1 was 2.9 μM. Inhibition of CYP1A2 and CYP2E1 was not increased by preincubation with ethambutol and β-nicotinamideadenine dinucleotide phosphate (NADPH), suggesting that the ethambutol-induced CYP inhibition may not be metabolism-dependent. Kinetic analysis showed that the inhibition of CYP1A2 and CYP2E1 by ethambutol was best fit to a competitive inhibition model. Formation of 1-methylxanthene and 1,3-dimethyluric acid from theophylline in HLM was decreased to 47% and 36%, respectively, by 3.0 μM ethambutol, which is comparable to its IC₅₀ value against CYP1A2. Considering its maximal plasma concentrations of ∼10 μM and long half-life of ∼22 h, our findings raise the possibility that ethambutol causes significant DDIs in clinical situations with drugs with narrow therapeutic index, such as theophylline, in clinical situations.     

Diarylheptanoid 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene from Curcuma comosa Roxb. protects retinal pigment epithelial cells against oxidative stress-induced cell death

Chronic exposure to oxidative stress causes damage to retinal pigment epithelial cells which may lead to the development of age-related macular degeneration, the major cause of vision loss in humans. Anti-oxidants provide a natural defense against retinal cell damage. The present study was designed to evaluate the potential anti-oxidant activity and protective effect of two diarylheptanoids isolated from a medicinal herb Curcuma comosa; 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound A), and 1,7-diphenyl-4(E),6(E)-heptadien-3-ol (compound B) against oxidative stress (H₂O₂)-induced human retinal pigment epithelial (APRE-19) cell death. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated that the anti-oxidant activity (IC₅₀) of compound A was similar to that of vitamin C. Pre-treatment of ARPE-19 cells with 20 μM compound A for 4 h afforded greater protection against the insult from 500 μM H₂O₂, compared to a similar protection period for compound B. Compound A lowered H₂O₂-induced lipid peroxidation, malondialdehyde formation and intracellular reactive oxygen species. Furthermore, compound A ameliorated the H₂O₂-induced decrease in anti-oxidant enzyme activities and subsequent apoptotic cell death in ARPE-19 cells in a dose and time-dependent manner. These results suggest that compound A protects ARPE-19 cells against oxidative stress, in part, by enhancing several anti-oxidant defense mechanisms. Therefore, compound A may have therapeutic potential for diseases associated with oxidative stress, particularly degenerative retinal diseases.     

New CYP1 genes in the frog Xenopus (Silurana) tropicalis: induction patterns and effects of AHR agonists during development

The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 μM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 μM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.     

Influence of GSTs, CYP2E1 and mEH polymorphisms on 1, 3-butadiene-induced micronucleus frequency in Chinese workers

1,3-butadiene (BD) has been classified as a human carcinogen, however, the relationship between chromosomal damage and its metabolic polymorphisms is not clear. The present study used the CBMN assay to detect chromosomal damage in the peripheral lymphocytes of 166 exposed workers and 41 non-exposed healthy individuals. PCR and PCR-RFLP were applied to detect GSTT1, GSTM1, CYP2E1 c1c2 and mEH Tyr113His, His139Arg polymorphisms. The results demonstrated that the micronucleus (MN) frequency of the exposed workers was significantly higher than controls (P < 0.01). Among the exposed workers, the individuals with high BD exposures are more susceptible to chromosomal damage than those with low exposures (FR = 1.30, 95% CI 1.14-1.53; P < 0.05). Gender-difference was also found in our study: males got lower micronucleus frequency than females. Workers who carried the genotypes of GSTM1 (+), CYP2E1 (c1c2/c2c2) and mEH intermediate (I) group had significantly higher MN frequency than those carrying the genotypes of GSTM1 (−) (FR = 1.29, 95% CI 1.05-1.59; P < 0.05), CYP2E1 (c1c1) (FR = 1.55, 95% CI 1.24-1.93; P < 0.01) or mEH high (H) group (FR = 1.57, 95% CI 1.08-2.34; P < 0.05), respectively. Our data indicated that the current BD exposure level could cause significantly higher MN frequency in workers than controls. Polymorphisms of GSTM1, CYP2E1 and mEH are susceptible to altered chromosome damage.     

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC₅₀ value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c_max at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c_max. The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K_i values were determined. All K_i values exceeded c_max by at least a factor of 10-fold. According to FDA regulations 1 > c_max/K_i > 0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.     

N-Nitrosodiethylamine genotoxicity in primary rat hepatocytes: Effects of cytochrome P450 induction by phenobarbital

Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1 mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21-105 μg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21-21 μg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ∼6-fold and ∼2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105 μg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.     

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes

Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K_i value (0.155 μM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K_i = 0.0790 and 0.148 μM, respectively) compared with CYP1A1 (K_i = 0.541 μM). Δ⁹-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K_i = 2.47-7.54 μM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Δ⁹-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k_inact/K_I value (540 l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.     

Distinct interactions of 2′- and 3′-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the adenylyl cyclase toxin of Bacillus anthracis, edema factor

Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. B. anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study [Taha HM, Schmidt J, Göttle M, Suryanarayana S, Shen Y, Tang WJ, et al. Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2′(3′)-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides. Mol Pharmacol 2009;75:693-703] we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (K_i values in the 0.1-5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′- and 3′-MANT-isomers can bind to EF. The aim of the present study was to examine the effects of defined 2′- and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxy-isomers and the 2′(3′)-racemates. K_i values of MANT-nucleotides for inhibition of catalysis correlated with K_d values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′- and 3′-MANT-isomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions.     

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimuriumumu strains expressing human cytochrome P450 and N-acetyltransferase

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1 nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.     

A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Substances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumigatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC₅₀ = 3 μM) > 2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone (termed FUD-8, IC₅₀ = 20 μM) > 3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (IC₅₀ = 139 μM). Interestingly, these compounds also significantly suppressed the gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8 at 2.5 mg/kg showed significant, 47.9–51.7%, inhibition stronger than that of prednisolone at 10 mg/kg (41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory activity.     

Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction

Epidemiological studies indicated an enhancement of cigarette smoke-induced carcinogenicity, including hepatocellular carcinoma, by arsenic. We believe that arsenic will enhance the expression of hepatic CYP2A enzyme and NNK metabolism (a cigarette smoke component), thus its metabolites, and carcinogenic DNA adducts. Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential.     

Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid,dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

Trichloroethylene (TCE, CAS 79-01-6) is a widely used industrial chemical, and a common environmental pollutant. TCE is a well-known carcinogen in rodents and is classified as “probably carcinogenic to humans”. Several analytical methods have been proposed for detection of TCE metabolites in biological media utilizing derivatization-free techniques; however, none of them is suitable for simultaneous detection of both oxidative metabolites and glutathione conjugates of TCE in small volume biological samples. Here, we report a new combination of methods for assessment of major TCE metabolites: dichloroacetic acid (DCA), trichloroacetic acid (TCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG). First, DCA and TCA were extracted with ether. Second, the remaining aqueous fraction underwent solid phase extraction for DCVC and DCVG. Then, DCA and TCA were measured by hydrophilic interaction liquid chromatography ion exchange negative electrospray ionization tandem mass spectrometry, while DCVC and DCVG were measured by reverse phase positive electrospray ionization tandem mass spectrometry. This method was applied successfully to measure all 4 TCE metabolites in as little as 50 μl of serum from mice orally exposed to TCE (2100 mg/kg, 2 h). Serum concentrations (mean ± standard deviation) of the TCE metabolites obtained with this method are comparable or equivalent to those previously reported in the literature: DCA, 0.122 ± 0.014 nmol/ml (limit of detection: 0.01 nmol/ml); TCA, 256 ± 30 nmol/ml (0.4 nmol/ml); DCVG, 0.037 ± 0.015 nmol/ml (0.001 nmol/ml); DCVC, 0.0024 ± 0.0009 nmol/ml (0.001 nmol/ml). This method opens new opportunities to increase throughput and decrease number of animals required for mechanistic studies on TCE in rodents.