Catechol Metabolites Research Articles

A DFT Study on the Kinetics of HOO•, CH3OO•, and O2•- Scavenging by Quercetin and Flavonoid Catecholic Metabolites.

Reaction kinetics have been theoretically examined to ascertain the potency of quercetin (Q) and flavonoid catecholic metabolites 1-5 in the inactivation of HOO•, CH3OO•, and O2•- under physiological conditions. In lipidic media, the koverallTST/Eck rate constants for the proton-coupled electron transfer (PCET) mechanism indicate the catecholic moiety of Q and 1-5 as the most important in HOO• and CH3OO• scavenging. 5-(3,4-Dihydroxyphenyl)-γ-valerolactone (1) and alphitonin (5) are the most potent scavengers of HOO• and CH3OO•, respectively. The koverallMf rate constants, representing actual behavior in aqueous media, reveal Q as more potent in the inactivation of HOO• and CH3OO• via single electron transfer (SET). SET from 3-O- phenoxide anion of Q, a structural motif absent in 1-5, represents the most contributing reaction path to overall activity. All studied polyphenolics have a potency of O2•- inactivation via a concerted two-proton-coupled electron transfer (2PCET) mechanism. The obtained results indicate that metabolites with notable radical scavenging potency, and more bioavailability than ingested flavonoids, may contribute to human health-promoting effects ascribed to parent molecules.

3-OH Phloretin Inhibits High-Fat Diet-Induced Obesity and Obesity-Induced Inflammation by Reducing Macrophage Infiltration into White Adipose Tissue.

Phloretin and its glycoside phlorizin have been reported to prevent obesity induced by high-fat diet (HFD), but the effect of 3-OH phloretin, a catechol metabolite of phloretin, has not been investigated. In this study, we investigated the anti-obesity effects of phloretin and 3-OH phloretin in HFD-fed mice. The body weight gain induced by HFD was more inhibited by administration of 3-OH phloretin than by phloretin. The increases in fat mass, white adipose tissue (WAT) weight, adipocyte size, and lipid accumulation by HFD were also remarkably inhibited by 3-OH phloretin and, to a lesser extent, by phloretin. The HFD-induced upregulation of chemokines and pro-inflammatory cytokines was suppressed by 3-OH phloretin, preventing M1 macrophages from infiltrating into WAT and thereby reducing WAT inflammation. 3-OH phloretin also showed a more potent effect than phloretin on suppressing the expression of adipogenesis regulator genes, such as PPARγ2, C/EBPα, FAS, and CD36. Fasting blood glucose and insulin levels increased by HFD were diminished by the administration of 3-OH phloretin, suggesting that 3-OH phloretin may alleviate obesity-induced insulin resistance. These findings suggested that 3-OH phloretin has the potential to be a natural bioactive compound that can be used in the prevention or treatment of obesity and insulin resistance.

Zearalenone and its metabolite exposure directs oestrogen metabolism towards potentially carcinogenic metabolites in human breast cancer MCF-7 cells.

Zearalenone (ZEN) is produced by Fusarium species contaminating various agriculture crops. In this study, the effects of ZEN and its metabolites α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL) on the formation of carcinogenic oestrogen-catechols in MCF-7 cells were investigated. To assess the effects of mycoestrogens on the activity of cytochrome P450 1A1 and CYP1B1, the rate of ethoxyresorufin O-deethylation (EROD-assay) was measured. The effects of mycoestrogens on the expression of CYP 1A1, CYP 1B1, aryl-hydrocarbon receptor (AhR), and oestrogen receptor alpha (ERα) were determined by qPCR. The catechol-O-methyltransferase (COMT) activity was measured as the ratio of the methoxy metabolites of oestradiol. Results show that mycoestrogens inhibited significantly the CYP1-dependent EROD activities. In the presence of selective inhibitors, mycoestrogens reduced CYP 1A1 and enhanced CYP 1B1 activity. Quantitative PCR analyses demonstrated the upregulation of AhR and confirmed the selective effect of mycoestrogens on CYP1 expression levels and the decline of the CYP 1A1/CYP 1B1 ratio. Mycoestrogens increased the ratio of 4-MeOE to 2-MeOE2 formation significantly (P < 0.05). Our results suggest that the tested mycoestrogens increase the production of CYP1B1-mediated oestrogen catechol metabolites, directing the biotransformation of E2 towards 4-OHE2, which has been identified earlier as a crucial factor in oestrogen-induced tumour initiation.

Abstract 12954: A Genotype-Phenotype Link Between Cardiac Monoamine Oxidase Activity and Arrhythmogenic Potential Using a Translational Approach

Introduction: Monoamine oxidase (MAO) has emerged as an intriguing drug target for heart disease because of the toxicity of the oxidative catechol metabolites produced. Our group has recently shown that a high rate of MAO activity in atrial myocardium is associated with a greater incidence of postoperative atrial fibrillation (POAF) following coronary artery bypass graft surgery (CABG). Hypothesis: Cardiac MAO deficiency lowers arrhythmogenic potential. Methods and Results: In a cohort of age-matched CABG patients (N=288, 26% female, 20% African-American), we performed DNA-sequencing analysis of maoA,B while assessing maximal MAO-A and -B enzyme activity with norepinephrine (NE) in fresh atrial tissue from a subset (N=110) of these patients. Patients homozygous for maoA synonymous variant rs1800464, and maoB intron variant rs5905512, had lower NE-supported MAO activity ( Figure 1 , 0 = ref, 1=het, 2=homoz) and lower incidence of POAF (OR 0.6 ± 0.1 and 0.9 ± 0.02, respectively). Mice with cardiomyocyte-specific MAO-A deficiency (cMAOA -/- ) had 2-fold lower rates of epinephrine/caffeine-induced ventricular tachycardia (VT) compared with wild-type ( Figure 2 , *P&lt;0.05). Moreover, cardiomyocytes from cMAOA -/- mice had increased fractional shortening, faster relaxation and shorter Ca 2+ transient duration (P&lt;0.05). Alterations in Ca 2+ handling in the cMAOA -/- mice were accompanied by decreased epinephrine-induced phosphorylation of CamKII and ryanodine receptor (P&lt;0.05). Conclusions: Collectively, these findings suggest that decreasing MAO activity lowers arrhythmogenic potential in the heart, and that cardiac MAO may be a therapeutic target for POAF.

Identification of Possible Salivary Metabolic Biomarkers and Altered Metabolic Pathways in South American Patients Diagnosed with Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) represents 90% of oral malignant neoplasms. The search for specific biomarkers for OSCC is a very active field of research contributing to establishing early diagnostic methods and unraveling underlying pathogenic mechanisms. In this work we investigated the salivary metabolites and the metabolic pathways of OSCC aiming find possible biomarkers. Salivary metabolites samples from 27 OSCC patients and 41 control individuals were compared through a gas chromatography coupled to a mass spectrometer (GC-MS) technique. Our results allowed identification of pathways of the malate-aspartate shuttle, the beta-alanine metabolism, and the Warburg effect. The possible salivary biomarkers were identified using the area under receiver-operating curve (AUC) criterion. Twenty-four metabolites were identified with AUC > 0.8. Using the threshold of AUC = 0.9 we find malic acid, maltose, protocatechuic acid, lactose, 2-ketoadipic, and catechol metabolites expressed. We notice that this is the first report of salivary metabolome in South American oral cancer patients, to the best of our knowledge. Our findings regarding these metabolic changes are important in discovering salivary biomarkers of OSCC patients. However, additional work needs to be performed considering larger populations to validate our results.

Alternaria Mycotoxins: An Overview of Toxicity, Metabolism, and Analysis in Food.

The genus Alternaria is widely distributed in the environment. Numerous species of the genus Alternaria can produce a variety of toxic secondary metabolites, called Alternaria mycotoxins. In this review, natural occurrence, toxicity, metabolism, and analytical methods are introduced. The contamination of these toxins in foodstuffs is ubiquitous, and most of these metabolites present genotoxic and cytotoxic effects. Moreover, Alternaria toxins are mainly hydroxylated to catechol metabolites and combined with sulfate and glucuronic acid in in vitro arrays. A more detailed summary of the metabolism of Alternaria toxins is presented in this work. To effectively detect and determine the mycotoxins in food, analytical methods with high sensitivity and good accuracy are also reviewed. This review will guide the formulation of maximum residue limit standards in the future, covering both toxicity and metabolic mechanism of Alternaria toxins.

3,3'-Dichlorobiphenyl Is Metabolized to a Complex Mixture of Oxidative Metabolites, Including Novel Methoxylated Metabolites, by HepG2 Cells.

3,3'-Dichlorobiphenyl (PCB 11) is a byproduct of industrial processes and detected in environmental samples. PCB 11 and its metabolites are present in human serum, and emerging evidence demonstrates that PCB 11 is a developmental neurotoxicant. However, little is known about the metabolism of PCB 11 in humans. Here, we investigated the metabolism of PCB 11 and the associated metabolomics changes in HepG2 cells using untargeted high-resolution mass spectrometry. HepG2 cells were exposed for 24 h to PCB 11 in DMSO or DMSO alone. Cell culture media were analyzed with ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Thirty different metabolites were formed by HepG2 cells exposed to 10 μM PCB 11, including monohydroxylated, dihydroxylated, methoxylated-hydroxylated, and methoxylated-dihydroxylated metabolites and the corresponding sulfo and glucuronide conjugates. The methoxylated PCB metabolites were observed for the first time in a human-relevant model. 4-OH-PCB 11 (3,3'-dichlorobiphenyl-4-ol) and the corresponding catechol metabolite, 4,5-di-OH-PCB 11 (3',5-dichloro-3,4-dihydroxybiphenyl), were unambiguously identified based on liquid and gas chromatographic analyses. PCB 11 also altered several metabolic pathways, in particular vitamin B6 metabolism. These results demonstrate that complex PCB 11 metabolite profiles are formed in HepG2 cells that warrant further toxicological investigation, particularly since catechol metabolites are likely reactive and toxic.

Annexin A1 accounts for an anti-inflammatory binding target of sesamin metabolites

Sesamin [(7α,7′α,8α,8′α)-3,4:3′,4′-bis(methylenedioxy)-7,9′:7′,9-diepoxylignane] is a major lignan in sesame seeds. Sesamin is converted to the catechol metabolite, SC1 [(7α,7′α,8α,8′α)-3′,4′-methylenedioxy-7,9′:7′,9-diepoxylignane-3,4-diol] with anti-inflammatory effects after oral administration. However, its molecular target remains unknown. Analysis using high-performance affinity nanobeads led to the identification of annexin A1 (ANX A1) as an SC1-binding protein. SC1 was found to bind to the annexin repeat 3 region of ANX A1 with a high-affinity constant (Kd = 2.77 μmol L−1). In U937 cells, SC1 exhibited an anti-inflammatory effect dependent on ANX A1. Furthermore, administration of sesamin or SC1 attenuated carbon tetrachloride-induced liver damage in mice and concurrently suppressed inflammatory responses dependent on ANX A1. The mechanism involved SC1-induced ANX A1 phosphorylation at serine 27 that facilitates extracellular ANX A1 release. Consequently, the ANX A1 released into the extracellular space suppressed the production of tumor necrosis factor α. This study demonstrates that ANX A1 acts as a pivotal target of sesamin metabolites to attenuate inflammatory responses.

Sesame lignans reduce LDL oxidative susceptibility by downregulating the platelet-activating factor acetylhydrolase.

Low-density lipoprotein (LDL) oxidative susceptibility is recognized as a risk factor for atherosclerosis. We previously reported that the ingestion of a supplement containing sesame lignans (sesamin/episesamin) for 4 weeks reduced LDL oxidative susceptibility in humans. To elucidate the mechanisms underlying this observation, 12-week-old New Zealand White rabbits were fed a fat/cholesterol-enriched diet (100 g/day) for 6 weeks followed by oral administration of vehicle (control) or sesame lignans (50 mg/kg) for 4 weeks with the fat/cholesterol-enriched diet. The results showed that the ingestion of sesame lignans prolonged LDL oxidation lag time, regardless of the existence of the anti-oxidative catechol metabolite of sesamin/episesamin in LDL. Plasma platelet-activating factor acetylhydrolase (PAF-AH) activity was significantly reduced by sesame lignans. The prolongation of LDL oxidation lag time was abolished by the addition of a PAF-AH inhibitor. The expression level of pro-inflammatory cytokines and macrophage infiltration observed in the liver following the feeding of the fat/cholesterol-enriched diet were also significantly reduced by sesame lignans. These results indicate that sesame lignans reduce LDL oxidative susceptibility by downregulating plasma PAF-AH activity via the reduction of inflammation in the liver induced by fat/cholesterol-enriched diets.

Effects of the Catechol and Methoxy Metabolites of 17β-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2β, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2β metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2β metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2β metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2β metabolite-induced NO production. E2β and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-β antagonist) inhibited the rises in NOx levels stimulated by E2β and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (β2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2β, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2β metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy.

The impact of Catechol-O-methyl transferase knockdown on the cell proliferation of hormone-responsive cancers

Estrogen (E2) plays a central role in the development and progression of hormone-responsive cancers. Estrogen metabolites exhibit either stimulatory or inhibitory roles on breast and prostate cells. The catechol metabolite 4-hydroxyestradiol (4-OHE2) enhances cell proliferation, while 2-methoxyestradiol (2 ME) possesses anticancer activity. The major metabolizing enzyme responsible for detoxifying the deleterious metabolite 4-OHE2 and forming the anticancer metabolite 2 ME is Catechol-O-Methyl Transferase (COMT). The current work investigated the relationship between the expression level of COMT and the cell proliferation of hormone-responsive cancers. The results showed that COMT silencing enhanced the cell proliferation of ER-α positive cancer cells MCF-7 and PC-3 but not the cells that lack ER-α expression as MDA-MB231 and DU-145. The data generated from our study provides a better understanding of the effect of COMT on critical signaling pathways involved in the development and progression of breast cancer (BC) and prostate cancer (PC) including ER-α, p21cip1, p27kip1, NF-κB (P65) and CYP19A1. These findings suggest that COMT enzyme plays a tumor suppressor role in hormone receptor-positive tumors which opens the door for future studies to validate COMT expression as a novel biomarker for the prediction of cancer aggressiveness and treatment efficacy.

Genome-Wide Characterization of the Fur Regulatory Network Reveals a Link between Catechol Degradation and Bacillibactin Metabolism in Bacillus subtilis.

The ferric uptake regulator (Fur) is the global iron biosensor in many bacteria. Fur functions as an iron-dependent transcriptional repressor for most of its regulated genes. There are a few examples where holo-Fur activates transcription, either directly or indirectly. Recent studies suggest that apo-Fur might also act as a positive regulator and that, besides iron metabolism, the Fur regulon might encompass other biological processes such as DNA synthesis, energy metabolism, and biofilm formation. Here, we obtained a genomic view of the Fur regulatory network in Bacillus subtilis using chromatin immunoprecipitation sequencing (ChIP-seq). Besides the known Fur target sites, 70 putative DNA binding sites were identified, and the vast majority had higher occupancy under iron-sufficient conditions. Among the new sites detected, a Fur binding site in the promoter region of the catDE operon is of particular interest. This operon, encoding catechol 2,3-dioxygenase, is critical for catechol degradation and is under negative regulation of CatR and YodB. These three repressors (Fur, CatR, and YodB) function cooperatively to regulate the transcription of catDE, with Fur functioning as a sensor of iron limitation and CatR as the major sensor of catechol stress. Genetic analysis suggests that CatDE is involved in metabolism of the catecholate siderophore bacillibactin, particularly when bacillibactin is constitutively produced and accumulates intracellularly, potentially generating endogenous toxic catechol derivatives. This study documents a role for catechol degradation in bacillibactin metabolism and provides evidence that catechol 2,3-dioxygenase can detoxify endogenously produced catechol substrates in addition to its more widely studied role in biodegradation of environmental aromatic compounds and pollutants.IMPORTANCE Many bacteria synthesize high-affinity iron chelators (siderophores). Siderophore-mediated iron acquisition is an efficient and widely utilized strategy for bacteria to meet their cellular iron requirements. One prominent class of siderophores uses catecholate groups to chelate iron. B. subtilis bacillibactin, structurally similar to enterobactin (made by enteric bacteria), is a triscatecholate siderophore that is hydrolyzed to monomeric units after import to release iron. However, the ultimate fates of these catechol compounds and their potential toxicities have not been defined previously. We performed genome-wide identification of Fur binding sites in vivo and uncovered a connection between catechol degradation and bacillibactin metabolism in B. subtilis Besides its role in the detoxification of environmental catechols, the catechol 2,3-dioxygenase encoded by catDE also protects cells from intoxication by endogenous bacillibactin-derived catechol metabolites under iron-limited conditions. These findings shed light on the degradation pathway and precursor recycling of the catecholate siderophores.

Biosynthesis of redox-active metabolites in response to iron deficiency in plants.

Iron is an essential but poorly bioavailable nutrient due to its low solubility, especially in alkaline soils. Here, we describe the discovery of a novel redox-active catecholic metabolite, termed sideretin, which derives from the coumarin fraxetin, and is the primary molecule exuded by Arabidopsis thaliana roots in response to iron deficiency. We have identified two enzymes that complete the biosynthetic pathway of fraxetin and sideretin. Chemical characterization of fraxetin and sideretin, and biological assays with pathway mutants, suggest that these coumarins are critical for iron nutrition in A. thaliana. Further, we show that sideretin production occurs in eudicot species only distantly related to A. thaliana. Untargeted metabolomics of the root exudates of various eudicots revealed production of structurally diverse redox-active molecules in response to iron deficiency. Our results indicate that secretion of small-molecule reductants by roots may be a widespread and previously underappreciated component of reduction-based iron uptake.

Inhibitory effect of catecholic colonic metabolites of rutin on fatty acid hydroperoxide and hemoglobin dependent lipid peroxidation in Caco-2 cells.

To determine the preventive effect of dietary rutin on oxidative damages occurring in the digestive tract, 13-hydroperoxyoctadecadienoic acid and hemoglobin were exposed to Caco-2 intestinal cells after the pretreatment with colonic rutin metabolites. Among four catechol-type metabolites, quercetin and 3,4-dihydroxytoluene exerted significant protection on 13-hydroperoxyoctadecadienoic and hemoglobin-dependent lipid peroxidation of this epithelial cell. Compared with quercetin, a much lower concentration allowed 3,4-dihydroxytoluene to maximize the protective effect, though it needed a longer pre-incubation period. Neither quercetin nor 3,4-dihydroxytoluene affected the expression of peroxiredoxin-6 protein, which comprises the cellular antioxidant defense system. It is concluded that 3,4-dihydroxytoluene is a plausible rutin colonic metabolite that can suppress oxidative damages of intestinal epithelial cells by directly inhibiting lipid peroxidation. This result may illuminate the preventive role of dietary rutin against colorectal cancer incidence in relation to the consumption of red and processed meat.

Enantiopurity and absolute configuration determination of arene cis-dihydrodiol metabolites and derivatives using chiral boronic acids.

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis-1,2-diol metabolites, including boronate formation, using racemic or enantiopure (+) and (-)-2-(1-methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono- and poly-cyclic arene cis-dihydrodiol, cyclohex-2-en-1-one cis-diol, heteroarene cis/trans-2,3-diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2-[1-methoxy-2,2-dimethylpropyl]phenyl boronic acid (MDPBA) and 2-[1-methoxy-1-phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis-diol metabolites, are presented.

Antimicrobial metabolites from Saraca asoca impairs the membrane transport system and quorum-sensing system in Pseudomonas aeruginosa.

This study was conducted to explore the antimicrobial mechanism of metabolites from Saraca asoca (SA1) using differential proteomics and metabolic profile of Pseudomonas aeruginosa after treatment with effective sub-MIC dose of 312µg/mL. SA1 fraction was found to contain antibacterial metabolites catechol, protocatechuic acid, and epigallocatechin gallate. Proteome analysis revealed 33 differentially expressed proteins after SA1 treatment. Protein network analysis showed that SA1 treatment upregulated the DNA topological and metabolic processes. Furthermore, it revealed that T2SS, cellular component biogenesis, and response to chemical stimuli were inhibited by SA1 treatment, supported by down-regulated Na+/H+ antiporter, SdeX, ompK, and trbD proteins. Statistical analysis of mass data revealed the altered level of 20 metabolites includes HSLs, PQS, rhamnolipid, and pyocyanin. Proteome and metabolome results showed that treatment impaired cell membrane functions and quorum-sensing system. It was further confirmed by increased MDA (3.95 fold), and rhamnolipids (4.3 fold) production and, therefore, oxidative stress (36.9%) after SA1 treatment.

Studies on Para-Methoxymethamphetamine (PMMA) Metabolite Pattern and Influence of CYP2D6 Genetics in Human Liver Microsomes and Authentic Samples from Fatal PMMA Intoxications.

Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans; for this purpose, we used human liver microsomes (HLMs) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism by using genotyped HLMs isolated from CYP2D6 poor, population-average, and ultrarapid metabolizers. In HLMs, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), whereas low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA), and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, whereas OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLMs was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was two to seven times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. The present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism.

Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol-induced proliferation of ovine uterine artery endothelial cells.

The catechol metabolites of 17β-oestradiol (E2 β), 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through β-adrenoceptors (β-ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2 - and 4-OHE2 -induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2 β and other β-AR signalling hormones (i.e. catecholamines). Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2 β- and catecholamine-induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17β-oestradiol, which induces proliferation via oestrogen receptor-β (ER-β), the catecholoestradiols mediate P-UAEC proliferation via β-adrenoceptors (β-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE2 and 4-OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE2 and 4-OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17β-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.

Hyperthermia Severely Affects the Vascular Effects of MDMA and Metabolites in the Human Internal Mammary Artery In Vitro.

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") is a recreational drug used worldwide for its distinctive psychotropic effects. Although important cardiovascular effects, such as increased blood pressure and heart rate, have also been described, the vascular effects of MDMA and metabolites and their correlation with hyperthermia (major side effect of MDMA) are not yet fully understood and have not been previously reported. This study aimed at evaluating the effects of MDMA and its main catechol metabolites, alpha-methyldopamine (α-MeDA), N-methyl-alpha-methyldopamine (N-Me-α-MeDA), 5-(glutathion-S-yl)-alpha-methyldopamine [5-(GSH)-α-MeDA] and 5-(glutathion-S-yl)-N-methyl-alpha-methyldopamine [5-(GSH)-N-Me-α-MeDA], on the 5-HT-dependent vasoactivity in normothermia (37°C) and hyperthermia (40°C) of the human internal mammary artery (IMA) in vitro. The results showed the ability of MDMA, α-MeDA and N-Me-α-MeDA to exert vasoconstriction of the IMA which was considerably higher in hyperthermic conditions (about fourfold for MDMA and α-MeDA and twofold for N-Me-α-MeDA). The results also showed that all the compounds may influence the 5-HT-mediated concentration-dependent response of IMA, as MDMA, α-MeDA and N-Me-α-MeDA behaved as partial agonists and 5-(GSH)-α-MeDA and 5-(GSH)-N-Me-α-MeDA as antagonists. In conclusion, MDMA abuse may imply a higher cardiovascular risk associated both to MDMA and its metabolites that might be relevant in patients with underlying cardiovascular diseases, particularly in hyperthermia.

Free radical scavenging potency of quercetin catecholic colonic metabolites: Thermodynamics of 2H+/2e− processes

Reaction energetics of the double (2H+/2e−), i.e., the first 1H+/1e− (catechol→ phenoxyl radical) and the second 1H+/1e− (phenoxyl radical→ quinone) free radical scavenging mechanisms of quercetin and its six colonic catecholic metabolites (caffeic acid, hydrocaffeic acid, homoprotocatechuic acid, protocatechuic acid, 4-methylcatechol, and catechol) were computationally studied using density functional theory, with the aim to estimate the antiradical potency of these molecules. We found that second hydrogen atom transfer (HAT) and second sequential proton loss electron transfer (SPLET) mechanisms are less energy demanding than the first ones indicating 2H+/2e− processes as inherent to catechol moiety. The Gibbs free energy change for reactions of inactivation of selected free radicals indicate that catecholic colonic metabolites constitute an efficient group of more potent scavengers than quercetin itself, able to deactivate various free radicals, under different biological conditions. They could be responsible for the health benefits associated with regular intake of flavonoid-rich diet.