Yoko Watanabe

Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor

The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes.

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

Abstract 1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined. 2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. 3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. 4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. 5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. 6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. 7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

Expression of Avian β-Defensin 3, an Antimicrobial Peptide, by Sperm in the Male Reproductive Organs and Oviduct in Chickens: An Immunohistochemical Study

The aim of this study was to determine whether chicken sperm express avian beta-defensin 3 (AvBD-3), an antimicrobial peptide, and retain it during the residence in the male and female reproductive tract. Immunocytochemistry to identify AvBD-3 was performed in the testis, epididymis, ductus deferens, ejaculated sperm, and utero-vaginal junction of the oviduct of inseminated hens. Reverse transcription-PCR analysis was also performed to confirm the synthesis of AvBD-3 by testicular cells and ejaculated sperm. The immunoreaction products for AvBD-3 were identified in the elongated spermatids attached to the seminiferous epithelium and in the sperm in the lumen of seminiferous tubules. Immunoreaction products were found in the sperm in the epididymal ducts and ductus deferens. In the ejaculated sperm, immunoreaction products were distributed in the midpiece and cephalic region of the tail of sperm, but not in the head. The AvBD-3 immunoreaction products were also identified in the sperm stored in the sperm storage tubules in the utero-vaginal junction. Reverse transcription-PCR analysis showed the expression of AvBD-3 mRNA in the testicular tissue, but not in the ejaculated sperm. These results suggest that AvBD-3 is synthesized by late stage of spermatids in the testis, and this molecule is retained by the sperm during the passage of male reproductive tract, and even in the sperm storage tubules of oviduct. The sperm AvBD-3 is assumed to play roles in protecting the sperm from the microbial infection and enable to survive in the male and female reproductive tracts.

Prediction of human metabolism of the sedative-hypnotic zaleplon using chimeric mice transplanted with human hepatocytes.

Abstract 1. Human chimeric mice (h-PXB mice) having humanized liver, constructed by transplantation of human hepatocytes, were evaluated as an experimental model for predicting human drug metabolism. Metabolism of zaleplon in h-PXB mice was compared with that in rat chimeric mice (r-PXB mice) constructed by transplantation of rat hepatocytes. 2. Zaleplon is metabolized to 5-oxo-zaleplon by aldehyde oxidase and to desethyl-zaleplon by cytochrome P450 (CYP3A4) in rat and human liver preparations. 3. Liver S9 fraction of h-PXB mice metabolized zaleplon to 5-oxo-zaleplon and desethyl-zaleplon in similar amounts. However, liver S9 fractions of r-PXB and control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice predominantly metabolized zaleplon to desethyl-zaleplon. 5-Oxo-zaleplon was detected as a minor metabolite. 4. Oxidase activity of h-PXB mouse liver cytosol toward zaleplon was about 10-fold higher than that of r-PXB or control mice. In contrast, activities for desethyl-zaleplon formation were similar in liver microsomes from these mice, as well as rat and human liver microsomes. 5. In vivo, the level of 5-oxo-zaleplon in plasma of h-PXB mice was about 7-fold higher than that in r-PXB or control mice, in agreement with the in vitro data. Thus, aldehyde oxidase in h-PXB mice functions as human aldehyde oxidase, both in vivo and in vitro. 6. In contrast, the plasma level of desethyl-zaleplon in r-PXB and control mice was higher than that in h-PXB mice. 7. These results suggest h-PXB mice with humanized liver could be a useful experimental model to predict aldehyde oxidase- and CYP3A4-mediated drug metabolism in humans.

Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans

Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1-C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1-C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively.

Effects of Bu-Zhong-Yi-Qi-Tang on hepatic drug-metabolizing enzymes and plasma tolbutamide concentration in rats.

ETHNOPHARMACOLOGICAL RELEVANCE Bu-Zhong-Yi-Qi-Tang (BT) is the dry powder derived from the aqueous extract of a mixture of 10 medicinal herbs. It is a traditional Chinese medicine being used for the treatment of various immune-related diseases. AIM OF THE STUDY To investigate the effect of BT on hepatic drug-metabolizing enzymes and its effect on plasma concentrations of tolbutamide, a substrate of CYP2C, in rats. MATERIALS AND METHODS EXP 1: Thirty-two male Wistar rats were divided into four groups. Rats were fed a control diet and a control diet containing 1, 2.5 and 5% (w/w) of BT, respectively, for eight weeks. The activities of the major CYP and Phase II conjugating enzymes in rat liver microsomes as well as the antioxidant system in rat liver were assessed. Exp 2: Male Wistar rats were fed a control diet or a control diet containing 2.5% of BT, respectively, for eight weeks. A single 20-mg/kg oral dose of tolbutamide was then administered to each rat. Plasma samples were collected from each rat at 0.5, 1, 2, 4 and 8h after dosing. The concentrations of tolbutamide and glucose level in plasma were determined by high-performance liquid chromatography-mass spectrometer (HPLC/MS) and enzymatic method, respectively. RESULTS Significant decrease in microsomal CYP2C-catalyzed diclofenac 4-hydroxylation in the liver of rats fed the BT diet was observed. Increased UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) activities were also observed in the liver of rats fed the diet containing 2.5 and 5% of BT. Immunoblot analyses also showed decreases of CYP2C11 proteins in the liver of BT fed rats. In addition, rats fed the 2.5% BT diet for eight weeks had no effects on the disposition of tolbutamide and reduction of glucose level in plasma after orally administered of tolbutamide. CONCLUSIONS Rats fed the BT diet for eight weeks may decrease CYP2C enzyme activity and protein expression and increase Phase II conjugating enzyme activities in liver. However, BT may not affect the disposition and efficacy of tolbutamide.

Cytochrome P450-inhibitory activity of parabens and phthalates used in consumer products.

The in vitro cytochrome P450 (CYP)-inhibitory effects of 11 parabens and 7 phthalates used in consumer products, as well as their hydrolytic metabolites, were investigated, using rat liver microsomes as an enzyme source. The effects on individual CYP isozymes were evaluated by assaying inhibition of activities towards specific substrates, i.e., ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), pentoxyresorufin O-dealkylase (PROD), 7-benzyloxy-4-trifluoromethylcoumarin dealkylase (BFCD), 7-methoxy-4-trifluoromethylcoumarin dealkylase (MFCD) and 7-ethoxy-4-trifluoromethylcoumarin dealkylase (EFCD) activities. These activities were dose-dependently inhibited, most potently by medium-side-chain parabens (C6-9) and phthalates (C4-6), and less potently by shorter- and longer-side-chain esters. The hydrolytic product of parabens, 4-hydroxybenzoic acid, was not inhibitory, while those of phthalates, phthalic acid monoesters, showed lower inhibitory activity than the parent phthalates. Parabens showed relatively potent inhibition of MFCD activity, considered to be mainly due to CYP2C, and phthalates showed relatively potent inhibition of PROD activity, considered to be mainly due to CYP2B.

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes.

1. When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP. 2. The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole. 3. The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats. 4. When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.

Metabolism of methiocarb and carbaryl by rat and human livers and plasma, and effect on their PXR, CAR and PPARα activities

The oxidative, reductive, and hydrolytic metabolism of methiocarb and the hydrolytic metabolism of carbaryl by liver microsomes and plasma of rats or humans were examined. The effects of the metabolism of methiocarb and carbaryl on their nuclear receptor activities were also examined. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide, and a novel metabolite, methiocarb sulfone were detected. Methiocarb sulfoxide was oxidized to the sulfone by liver microsomes and reduced back to methiocarb by liver cytosol. Thus, the interconversion between methiocarb and the sulfoxide was found to be a new metabolic pathway for methiocarb by liver microsomes. The product of methiocarb hydrolysis, which is methylthio-3,5-xylenol (MX), was also oxidized to sulfoxide form by rat liver microsomes. The oxidations were catalyzed by human flavin-containing monooxygenase isoform (FMO1). CYP2C19, which is a human cytochrome P450 (CYP) isoform, catalyzed the sulfoxidations of methiocarb and MX, while CYP1A2 also exhibited oxidase activity toward MX. Methiocarb and carbaryl were not enzymatically hydrolyzed by the liver microsomes, but they were mainly hydrolyzed by plasma and albumin to MX and 1-naphthol, respectively. Both methiocarb and carbaryl exhibited PXR and PPARα agonistic activities; however, methiocarb sulfoxide and sulfone showed markedly reduced activities. In fact, when methiocarb was incubated with liver microsomes, the receptor activities were decreased. In contrast, MX and 1-naphthol showed nuclear receptor activities equivalent to those of their parent carbamates. Thus, the hydrolysis of methiocarb and carbaryl and the oxidation of methiocarb markedly modified their nuclear receptor activities.