In vitro hepatic metabolism of the natural product quebecol.

Quebecol (2,3,3-tri-(3-methoxy-4-hydroxyphenyl)-1-propanol) is a polyphenolic compound, which is formed during maple syrup production from Acer spp. Quebecol bears structural similarities to the chemotherapy drug tamoxifen, which has led to synthesis of structural analogues and investigations into their pharmacological properties, however there are no reports on the hepatic metabolism of quebecol.This interest in therapeutic properties spurred us to investigate the in vitro microsomal Phase I and II metabolism of quebecol. We were unable to detect any P450 metabolites for quebecol in either human liver microsomes (HLM) or rat liver microsomes (RLM). In contrast we observed marked formation of three glucuronide metabolites in both RLM and HLM, suggesting that clearance via Phase II pathways is likely to predominate.To further understand the hepatic contribution to first-pass glucuronidation we have validated an HPLC method following FDA and EMA guidelines (selectivity, linearity, accuracy, and precision) to quantify quebecol in microsomes. In vitro enzyme kinetics were performed for quebecol glucuronidation by HLM including 8 concentrations from 5-30 µM. We determined a Michaelis-Menten constant (KM) of 5.1 µM, intrinsic clearance (Clint,u) of 0.038 ± 0.001 mL/min/mg, and maximum velocity (Vmax) of 0.22 ± 0.01 µmol/min/mg.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Tandem mass spectrometric analysis of novel caffeine scaffold-based bifunctional compounds for Parkinson's disease.

RATIONALE: Novel bifunctional compounds composed of a caffeine scaffold attached to nicotine (C8 -6-N), 1-aminoindan (C8 -6-I), or caffeine (C8 -6-C8 ) were designed as therapeutics or diagnostics for Parkinson's disease (PD). In order to probe their pharmacological and toxicological profile, an appropriate analytical method is required. The goal of this study is to establish a tandem mass spectrometric fingerprint for the development of quantitative and qualitative methods that will aid future assessment of the in vitro and in vivo absorption, distribution, metabolism, excretion (ADME) and pharmacokinetic properties of these lead bifunctional compounds for PD. METHODS: Accurate mass measurement was performed using a hybrid quadrupole orthogonal time-of-flight mass spectrometer while multistage MS/MS and MS3 analyses were conducted using a triple quadrupole linear ion trap mass spectrometer. Both instruments are equipped with an electrospray ionization (ESI) source and were operated in the positive ion mode. The source and compound parameters were optimized for all three tested bifunctional compounds. RESULTS: The MS/MS analysis indicates that the fragmentation of C8 -6-N and C8 -6-I is driven by the dissociation of the nicotine and 1-aminoindan moieties, respectively, but not caffeine. A significant observation in the MS/MS fragmentation of C8 -6-C8 suggests that a previously reported loss of acetaldehyde during caffeine dissociation is instead a loss of CO2 . CONCLUSIONS: The collision-induced tandem mass spectrometry (CID-MS/MS) analysis of these novel bifunctional compounds revealed compound-specific diagnostic product ions and neutral losses for all three tested bifunctional compounds. The established MS/MS fingerprint will be applied to the future development of qualitative and quantitative methods.

Residues His172 and Lys238 are Essential for the Catalytic Activity of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain L-1.

Maleylacetate reductase (PcpE), the last enzyme in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum L-1, catalyzes two consecutive reductive reactions, reductive dehalogenation of 2-chloromaleylacetate (2-CMA) to maleylacetate (MA) and subsequent reduction of MA to 3-oxoadipate (3-OXO). In each reaction, one molecule of NADH is consumed. To better understand its catalytic function, we undertook a structural model-based site-directed mutagenesis and steady-state kinetics study of PcpE. Our results showed that the putative catalytic site of PcpE is located in a positively charged solvent channel at the interface of the two domains and the binding of 2-CMA/MA involves seven basic amino acids, His172, His236, His237, His241 and His251, Lys140 and Lys238. Mutagenesis studies showed that His172 and Lys238 are essential for the catalytic activity of PcpE. However, the mutation of His236 to an alanine can increase the catalytic efficiency (k cat /K m ) of PcpE by more than 2-fold, implying that PcpE is still in an early stage of molecular evolution. Similar to tetrachlorobenzoquinone reductase (PcpD), PcpE is also inhibited by pentachlorophenol in a concentration-dependent manner. Furthermore, our studies showed that PcpE exhibits an extremely low but detectable level of alcohol dehalogenase activity toward ethanol and supports the notion that it is evolved from an iron-containing alcohol dehydrogenase.

Enterolactone glucuronide and ß-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment.

Evidence from preclinical and animal studies demonstrated an anticancer effect of flaxseed lignans, particularly enterolactone (ENL), against prostate cancer. However, extensive first-pass metabolism following oral lignan consumption results in their systemic availability primarily as glucuronic acid conjugates (ENL-Gluc) and their modest in vivo effects. To overcome the unfavorable pharmacokinetics and improve their effectiveness in prostate cancer, antibody-directed enzyme prodrug therapy (ADEPT) might offer a novel strategy to allow for restricted activation of ENL from circulating ENL-Gluc within the tumor environment. The anti-prostate-specific membrane antigen (PSMA) antibody D7 was fused with human ß-glucuronidase (hßG) via a flexible linker. The binding property of the fusion construct, D7-hßG, against purified or cell surface PSMA was determined by flow cytometry and Octet Red 384 system, respectively, with a binding rate constant, K d, of 2.5 nM. The enzymatic activity of D7-hßG was first tested using the probe, 4-methylumbelliferone glucuronide. A 3.8-fold greater fluorescence intensity was observed at pH 4.5 at 2 h compared with pH 7.4. The ability of D7-hßG to activate ENL from ENL-Gluc was tested and detected using LC-MS/MS. Enhanced generation of ENL was observed with increasing ENL-Gluc concentrations and reached 3613.2 ng/mL following incubation with 100 µM ENL-Gluc at pH 4.5 for 0.5 h. D7-hßG also decreased docetaxel IC50 value from 23 nM to 14.9 nM in C4-2 cells. These results confirmed the binding and activity of D7-hßG and additional in vitro investigation is needed to support the future possibility of introducing this ADEPT system to animal models.

Flavonols Protect Against UV Radiation-Induced Thymine Dimer Formation in an Artificial Skin Mimic.

PURPOSE: Exposure of skin to ultraviolet light has been shown to have a number of deleterious effects including photoaging, photoimmunosuppression and photoinduced DNA damage which can lead to the development of skin cancer. In this paper we present a study on the ability of three flavonols to protect EpiDerm™, an artificial skin mimic, against UV-induced damage. METHODS: EpiDerm™ samples were treated with flavonol in acetone and exposed to UVA (100 kJ/m(2) at 365 nm) and UVB (9000 J/m(2) at 310 nm) radiation. Secretion of matrix metalloproteinase-1 (MMP-1) and tumor necrosis factor-α (TNF-a) were determined by ELISA, cyclobutane pyrimidine dimers were quantified using LC-APCI-MS. RESULTS: EpiDerm™ treated topically with quercetin significantly decreased MMP-1 secretion induced by UVA (100 µM) or UVB (200 µM) and TNF-a secretion was significantly reduced at 100 µM quercetin for both UVA and UVB radiation. In addition, topically applied quercetin was found to be photostable over the duration of the experiment. EpiDerm™ samples were treated topically with quercetin, kaempferol or galangin (52 µM) immediately prior to UVA or UVB exposure, and the cyclobutane thymine dimers (T-T (CPD)) were quantified using an HPLC-APCI MS/MS method. All three flavonols significantly decreased T-T (CPD) formation in UVB irradiated EpiDerm™, however no effect could be observed for the UVA irradiation experiments as thymine dimer formation was below the limit of quantitation. CONCLUSIONS: Our results suggest that flavonols can provide protection against UV radiation-induced skin damage through both antioxidant activity and direct photo-absorption. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Ring substitution influences oxidative cyclisation and reactive metabolite formation of nordihydroguaiaretic acid analogues.

Nordihydroguaiaretic acid (NDGA) is a natural polyphenol with a broad spectrum of pharmacological properties. However, its usefulness is hindered by the lack of understanding of its pharmacological and toxicological pathways. Previously we showed that oxidative cyclisation of NDGA at physiological pH forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to an ortho-quinone likely mediates toxicological properties. NDGA analogues with higher propensity to cyclise under physiologically relevant conditions might have pharmacological implications, which motivated this study. We synthesized a series of NDGA analogues which were designed to investigate the structural features which influence the intramolecular cyclisation process and help to understand the mechanism of NDGA's autoxidative conversion to a dibenzocyclooctadiene lignan. We determined the ability of the NDGA analogues investigated to form dibenzocyclooctadienes and evaluated the oxidative stability at pH 7.4 of the analogues and the stability of any dibenzocyclooctadienes formed from the NDGA analogues. We found among our group of analogues the catechols were less stable than phenols, a single catechol-substituted ring is insufficient to form a dibenzocyclooctadiene lignan, and only compounds possessing a di-catechol could form dibenzocyclooctadienes. This suggests that quinone formation may not be necessary for cyclisation to occur and the intramolecular cyclisation likely involves a radical-mediated rather than an electrophilic substitution process. We also determined that the catechol dibenzocyclooctadienes autoxidised at comparable rates to the parent catechol. This suggests that assigning in vitro biological activity to the NDGA dibenzocyclooctadiene is premature and requires additional study.

Comparative pharmacokinetics of purified flaxseed and associated mammalian lignans in male Wistar rats.

Consumption of flaxseed lignans is associated with various health benefits; however, little is known about the bioavailability of purified lignans in flaxseed. Data on their bioavailability and hence pharmacokinetics (PK) are necessary to better understand their role in putative health benefits. In the present study, we conducted a comparative PK analysis of the principal lignan of flaxseed, secoisolariciresinol diglucoside (SDG), and its primary metabolites, secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL) in rats. Purified lignans were intravenously or orally administered to each male Wistar rat. SDG and its primary metabolites SECO, ED and EL were administered orally at doses of 40, 40, 10 and 10 mg/kg, respectively, and intravenously at doses of 20, 20, 5 and 1 mg/kg, respectively. Blood samples were collected at 0 (pre-dose), 5, 10, 15, 20, 30 and 45 min, and at 1, 2, 4, 6, 8, 12 and 24 h post-dosing, and serum samples were analysed. PK parameters and oral bioavailability of purified lignans were determined by non-compartmental methods. In general, administration of the flaxseed lignans SDG, SECO and ED demonstrated a high systemic clearance, a large volume of distribution and short half-lives, whereas administration of EL at the doses of 1 mg/kg (intravenously) and 10 mg/kg (orally administered) killed the rats within a few hours of dosing, precluding a PK analysis of this lignan. PK parameters of flaxseed lignans exhibited the following order: systemic clearance, SDG < SECO < ED; volume of distribution, SDG < SECO < ED; half-life, SDG < ED < SECO. The percentage of oral bioavailability was 0, 25 and < 1 % for SDG, SECO and ED, respectively.

UVA and UVB radiation-induced oxidation products of quercetin.

The flavonol quercetin is believed to provide protection against ultraviolet (UV) radiation-induced damage in plants. As part of our investigations into the potential for quercetin to protect skin against UV radiation-induced damage we have investigated the products of quercetin exposed to UV radiation in vitro. UVA (740 microW cm(-2) at 365 nm) or UVB (1300 microW cm(-2) at 310 nm) irradiation of quercetin in methanol results in a small conversion (less than 20%) to C-ring breakdown products over 11 h. When the triplet sensitizer benzophenone is added, greater than 90% conversion by UVA or UVB occurs within 1h. The major photoproducts from either UVA or UVB radiation are 2,4,6-trihydroxybenzaldehyde (1), 2-(3',4'-dihydroxybenzoyloxy)-4,6-dihydroxybenzoic acid (2) and 3,4-dihydroxyphenylethanol (3). Product 2 has previously been observed as a product of oxidative metabolism of quercetin, however products 1 and 3 appear to be the result of a unique UV-dependent pathway. In conclusion we have determined that quercetin undergoes slow decomposition to a mixture of C-ring-opened products, two of which to our knowledge have not been previously observed for quercetin decomposition, and that the presence of a triplet sensitizer greatly increases UV radiation-mediated quercetin decomposition. The presence of endogenous photosensitizers in the skin could potentially affect the UV stability of quercetin, suggesting that further study of quercetin for both its photoprotective properties and photostabilty in skin are warranted.

Inhibition of UVA and UVB radiation-induced lipid oxidation by quercetin.

The flavonol quercetin is believed to provide protection against ultraviolet (UV)-induced damage to plants. As part of our investigations into the potential for quercetin to protect skin against UV-induced damage, we have measured the ability of quercetin to inhibit UV-induced lipid peroxidation in vitro in liposomes. Quercetin, which absorbs UV radiation at 255 and 365 nm, was determined to be a stronger inhibitor of lipid oxidation induced by UVB (3.7 radicals scavenged per molecule) than by lipid oxidation induced by UVA (1.9 radicals scavenged per molecule). The values for inhibition of UVB-induced lipid oxidation by quercetin are comparable to those when 2,2'-azobis(2-amidinopropane) was used as an oxidizing system (four radicals scavenged per molecule). The protective effect of quercetin appears to be mainly the result of scavenging of UV-generated radical species, although this may be decreased slightly in the UVA as a result of its absorption at 365 nm.

Cloning, overexpression, purification, and characterization of the maleylacetate reductase from Sphingobium chlorophenolicum strain ATCC 53874.

The final enzyme in the pentachlorophenol (PCP) biodegradation pathway in Sphingobium chlorophenolicum is maleylacetate reductase (PcpE), which catalyzes the reductive dehalogenation of 2-chloromaleylacetate to maleylacetate and the subsequent reduction of malyelacetate to 3-oxoadipate. In this study, the pcpE gene was cloned from S. chlorophenolicum strain ATCC 53874 and overexpressed in Escherichia coli BL21-AI cells. The recombinant PcpE, purified to higher than 95% purity using affinity chromatography, exhibited optimal activity at pH 7.0. The kinetic parameters k(cat) and K(m) were 1.2 +/- 0.3 s(-1) and 0.09 +/- 0.04 mM, respectively, against maleylacetate under the optimal pH. In addition, the purified PcpE was able to restore PCP-degrading capability to S. chlorophenolicum strain ATCC 39723, implicating that there was no functional PcpE in the ATCC 39723 strain.

Oxidation of the lignan nordihydroguaiaretic acid.

The naturally occurring antioxidant lignan nordihydroguaiaretic acid has been used in traditional medicine to treat a variety of conditions and more recently has been investigated for its anticancer and antimicrobial properties. Nordihydroguaiaretic acid has also been shown to cause kidney toxicity in rats and there is evidence to suggest that chronic nordihydroguaiaretic acid consumption may cause liver toxicity in humans. Nordihydroguaiaretic acid likely undergoes biotransformation to a reactive quinone species, either an ortho-quinone or a para-quinone methide, which is responsible for its toxicity. In an effort to probe the toxicity of nordihydroguaiaretic acid, we oxidized nordihydroguaiaretic acid with both chemical and enzymatic systems and trapped the resultant products with glutathione. The nordihydroguaiaretic acid-glutathione adducts were compared with the products found when nordihydroguaiaretic acid was incubated in rat hepatic microsomes in the presence of glutathione. Glutathione metabolites consistent with ortho-quinone formation were the only oxidation products observed. Control experiments in microsomes however suggested that a majority of the nordihydroguaiaretic acid ortho-quinone glutathione adducts were formed as the result of nordihydroguaiaretic acid autoxidation. We measured the rate of this autoxidation process over a range of pH values and determined that the autoxidation reaction is base-catalyzed. We suggest that caution must be exercised when using nordihydroguaiaretic acid in experiments above pH 7.4 as relatively little of the parent compound may be left in incubations longer than 3 h.

AAPH-mediated antioxidant reactions of secoisolariciresinol and SDG.

Secoisolariciresinol (SECO ) is the major lignan found in flaxseed (Linum usitatissimum L.) and is present in a polymer that contains secoisolariciresinol diglucoside (SDG ). SECO, SDG and the polymer are known to have a number of health benefits, including reduction of serum cholesterol levels, delay in the onset of type II diabetes and decreased formation of breast, prostate and colon cancers. The health benefits of SECO and SDG may be partially attributed to their antioxidant properties. To better understand their antioxidant properties, SECO and SDG were oxidized using 2,2'-azobis(2-amidinopropane), an in vitro model of radical scavenging. The major lignan radical-scavenging oxidation products and their formation over time were determined. SDG was converted to four major products, which were the result of a phenoxyl radical intermediate. One of these products, a dimer of SDG, decomposed under the reaction conditions to form two of the other major products, and . SECO was converted to five major products, two of which were also the result of a phenoxyl radical intermediate. The remaining products were the result of an unexpected alkoxyl radical intermediate. The phenol oxidation products were stable under the reaction conditions, whereas two of the alcohol oxidation products decomposed. In general, only one phenol group on the lignans was oxidized, suggesting that the number of phenols per molecule may not predict radical scavenging antioxidant ability of lignans. Finally, SECO is a superior antioxidant to SDG, and it may be that the additional alcohol oxidation pathway contributes to its greater antioxidant ability.