Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

Isoform-Specific Quantification of Human Bitter Taste Receptor Transcripts Using Real-Time PCR Analysis.

Bitter taste receptors (TAS2Rs) are expressed by oral cavity cells in mammals and classically function as sensors for bitter compounds. There are 25 functional isoforms of human TAS2Rs, with individual bitter ligands. Each human TAS2R isoform is distributed in several tissues, such as the airway epithelia and gastrointestinal tract, and plays an important role in physiological functions. However, quantification of each isoform is difficult because of highly homologous sequences between some TAS2R isoforms. Therefore, differentiating the isoforms by their expression levels is suitable for clarifying the tissue-specific effects of bitter compounds. In this study, we developed a real-time quantitative PCR (qPCR) method to determine the expression of each TAS2R isoform. Using plasmid standards harboring each isoform, we confirmed that the current assay can quantify the gene expression of each isoform, with negligible interference from other isoforms. In addition, our methods can successfully discriminate between the mRNA expression of each isoform in human cell lines and tissues. Therefore, this qPCR method can successfully quantify the mRNA level of each TAS2R isoform. This method will contribute to a better understanding of the molecular mechanisms underlying the TAS2R ligand-activated signal transduction.

Species-Specific Activation of Transient Receptor Potential Ankyrin 1 by Phthalic Acid Monoesters.

Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC50 values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.

In vitro glucuronidation of bisphenol A in liver and intestinal microsomes: interspecies differences in humans and laboratory animals.

Bisphenol A (BPA) is an endocrine-disrupting chemical, and is predominantly metabolized into glucuronide in mammals. The present study was conducted in order to examine the hepatic and intestinal glucuronidation of BPA in humans and laboratory animals such as monkeys, dogs, rats, and mice in an in vitro system using microsomal fractions. Km, Vmax, and CLint values in human liver microsomes were 7.54 µM, 17.7 nmol/min/mg protein, and 2.36 mL/min/mg protein, respectively. CLint values in liver microsomes of monkey, dogs, rats, and mice were 1.5-, 2.4-, 1.7- and 8.2-fold that of humans, respectively. In intestinal microsomes, Km, Vmax, and CLint values in humans were 39.3 µM, 0.65 nmol/min/mg protein, and 0.02 mL/min/mg protein, respectively. The relative levels of CLint in monkey, dogs, rats, and mice to that of humans were 7.0-, 12-, 34-, and 29-fold, respectively. Although CLint values were higher in liver microsomes than in intestinal microsomes in all species, and marked species difference in the ratio of liver to intestinal microsomes was observed as follows: humans, 118; monkeys, 25; dogs, 23; rats, 5.9; mice, 33. These results suggest that the functional roles of UDP-glucuronosyltransferase (UGT) enzymes expressed in the liver and intestines in the metabolism of BPA extensively differ among humans, monkeys, dogs, rats, and mice.

Favipiravir biotransformation in liver cytosol: Species and sex differences in humans, monkeys, rats, and mice.

Favipiravir is an antiviral agent effective against several RNA viruses that is converted into an inactive oxidative metabolite (M1), mainly by aldehyde oxidase, in humans. In the present study, the biotransformation of favipiravir into M1 in male and female humans, monkeys, rats, and mice was examined in an in vitro system using liver cytosolic fractions. The kinetics for M1 formation followed the Michaelis-Menten model in all species. The Km , Vmax , and CLint values in humans were 602 µM, 466 pmol/min/mg protein, and 776 nl/min/mg protein in males, respectively, and 713 µM, 404 pmol/min/mg protein, and 567 nl/min/mg protein in females, respectively. Species differences in CLint values were monkeys > humans > mice > rats in both males and females, and the variations for males and females were 120- and 96-fold, respectively. Sex differences in CLint values were males > females in humans and mice, females > males in monkeys and rats, and marked variation (4.3-fold) was noted in mice. This suggests that the roles of aldehyde oxidase in the hepatic metabolism of favipiravir differ extensively depending on the species and sex, and this study will aid in the assessment of the antiviral activities of favipiravir against novel and/or variant viruses.

Wogonin glucuronidation in liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice.

Wogonin, one of the flavonoids isolated from Scutellaria baicalensis, exhibits some beneficial bioactivities, including anti-inflammatory and anticancer effects, and is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. In the present study, wogonin glucuronidation was examined in the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice using a kinetic analysis.The kinetics of wogonin glucuronidation by liver microsomes followed the biphasic model in all species examined. CLint values (x-intercept) based on v versus V/[S] plots were rats > humans ≈ monkeys > mice > dogs. The kinetics of intestinal microsomes fit the Michaelis-Menten model for humans, monkeys, rats, and mice and the substrate inhibition model for dogs. CLint values were rats > monkeys > mice > dogs > humans. The tissue dependence of CLint values was liver microsomes > intestinal microsomes for humans, dogs, and rats, and liver microsomes ≈ intestinal microsomes for monkeys and mice.These results demonstrated that the metabolic abilities of UGT enzymes toward wogonin in the liver and intestines markedly differ among humans, monkeys, dogs, rats, and mice, and suggest that species differences are closely associated with the biological effects of wogonin.

Regioselective glucuronidation of daidzein in liver and intestinal microsomes of humans, monkeys, rats, and mice.

Daidzein, one of the major soy isoflavones, has a number of beneficial bioactivities for human health. It is mainly metabolized into 7- and/or 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in mammals, including humans. The present study was conducted to examine the regioselective glucuronidation of daidzein at the 7- and 4'-hydroxyl groups in the liver and intestinal microsomes of humans, monkeys, rats, and mice. Daidzein glucuronidation activities at substrate concentrations of 1.0-200 µM were assessed, and Eadie-Hofstee plots were constructed. The kinetics for 7- and 4'-glucuronidation in the liver microsomes fit the Michaelis-Menten model, except for an atypical model for 7-glucuronidation in rats and a biphasic model for 4'-glucuronidation in monkeys. These kinetics in the intestinal microsomes followed the Michaelis-Menten model, except for a biphasic model for 7-glucuronidation in mice. The CLint values for 7-glucuronidation were in the order of monkeys (49) ≫ rats (5.3) > humans (1.0) > mice (0.7) for liver microsomes, and rats (2.4) ≥ monkeys (2.2) > humans (1.0) ≥ mice (0.8) for intestinal microsomes. On the other hand, the CLint values for 4'-glucuronidation were in the order of monkeys (4.0) > mice (1.0) ≈ humans (1.0) > rats (0.4) for liver microsomes, and humans (1.0) ≫ monkeys (0.08) ≥ mice (0.07) > rats (0.05) for intestinal microsomes. These results demonstrated that the metabolic abilities of UGT enzymes toward daidzein in the liver and intestines markedly differed among humans, monkeys, rats, and mice, and suggest that species and regioselective differences are closely associated with the bioactivities of soy isoflavones.

Glucuronidation of mono(2-ethylhexyl) phthalate in humans: roles of hepatic and intestinal UDP-glucuronosyltransferases.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP), which is an endocrine-disrupting chemical. In the present study, MEHP glucuronidation in humans was studied using recombinant UDP-glucuronosyltransferases (UGTs) and microsomes of the liver and intestine. Among the recombinant UGTs examined, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, and UGT2B7 glucuronidated MEHP. The kinetics of MEHP glucuronidation by UGT1A3, UGT1A7, UGT1A8, UGT1A10, UGT2B4, and UGT2B7 followed the Michaelis-Menten model, whereas that by UGT1A9 fit the negative allosteric model. CLint values were in the order of UGT1A9 > UGT2B7 > UGT1A7 > UGT1A8 ≥ UGT1A10 > UGT1A3 > UGT2B4. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model. Diclofenac (20 µM) and raloxifene (20 µM) decreased CLint values to 43 and 36 % that of native microsomes, respectively. The kinetics of MEHP glucuronidation by intestine microsomes fit the biphasic model. Diclofenac (150 and 450 µM) decreased CLint values to 32 and 13 % that of native microsomes for the high-affinity phase, and to 28 and 21 % for the low-affinity phase, respectively. Raloxifene (2.5 and 7.0 µM) decreased CLint values to 35 and 4.1 % that of native microsomes for the high-affinity phase, and to 48 and 53 % for the low-affinity phase, respectively. These results suggest that MEHP glucuronidation in humans is catalyzed by UGT1A3, UGT1A9, UGT2B4, and/or UGT2B7 in the liver, and by UGT1A7, UGT1A8, UGT1A9, UGT1A10, and/or UGT2B7 in the intestine, and also that these UGT isoforms play important and characteristic roles in the detoxification of DEHP.

Glucuronidation of 4-tert-octylphenol in humans, monkeys, rats, and mice: an in vitro analysis using liver and intestine microsomes.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the glucuronidation of 4-tOP in humans, monkeys, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of 4-tOP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and monkeys, and the biphasic model for rats and mice. The K m, V max, and CL int values of human liver microsomes were 0.343 µM, 11.6 nmol/min/mg protein, and 33.8 mL/min/mg protein, respectively. The kinetics of intestine microsomes followed the Michaelis-Menten model for humans, monkeys, and rats, and the biphasic model for mice. The K m, V max, and CL int values of human intestine microsomes were 0.743 µM, 0.571 nmol/min/mg protein, and 0.770 mL/min/mg protein, respectively. The CL int values estimated by Eadie-Hofstee plots were in the order of mice (high-affinity phase) (3.0) > humans (1.0) ≥ monkeys (0.9) > rats (high-affinity phase) (0.4) for liver microsomes, and monkeys (10) > mice (high-affinity phase) (5.6) > rats (1.4) > humans (1.0) for intestine microsomes. The percentages of the CL int values of intestine microsomes to liver microsomes were in the order of monkeys (27 %) > rats (high-affinity phase in liver microsomes) (7.9 %) > mice (high-affinity phase in liver and intestine microsomes) (4.2 %) > humans (2.3 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward 4-tOP markedly differ among species and imply that species differences are strongly associated with the toxicities of alkylphenols.

Hepatic glucuronidation of 4-tert-octylphenol in humans: inter-individual variability and responsible UDP-glucuronosyltransferase isoforms.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. The purpose of this study was to assess inter-individual variability in and the possible roles of UGT isoforms in hepatic 4-tOP glucuronidation in the humans. 4-tOP glucuronidation activities in the liver microsomes and recombinant UGTs of humans were assessed at broad substrate concentrations, and kinetics were analyzed. Correlation analyses between 4-tOP and diclofenac or 4-hydroxybiphenyl activities in pooled and individual human liver microsomes were also performed. Typical CLint values were 17.8 mL/min/mg protein for the low type, 25.2 mL/min/mg protein for the medium type, and 47.7 mL/min/mg protein for the high type. Among the recombinant UGTs (13 isoforms) examined, UGT2B7 and UGT2B15 were the most active of catalyzing 4-tOP glucuronidation. Although the K m values of UGT2B7 and UGT2B15 were similar (0.36 and 0.42 µM, respectively), the CLint value of UGT2B7 (6.83 mL/min/mg protein) >UGT2B15 (2.35 mL/min/mg protein). Strong correlations were observed between the glucuronidation activities of 4-tOP and diclofenac (a probe for UGT2B7) or 4-hydroxybiphenyl (a probe for UGT2B15) with 0.79-0.88 of Spearman correlation coefficient (r s) values. These findings demonstrate that 4-tOP glucuronidation in humans is mainly catalyzed by hepatic UGT2B7 and UGT2B15, and suggest that these UGT isoforms play important and characteristic roles in the detoxification of 4-tOP.

Analysis of glycols, glycol ethers, and other volatile organic compounds present in household water-based hand pump sprays.

The aim of this investigation is to clarify the types and concentrations of VOCs present in various commercial household water-based hand pump spray products used in Japan, and to estimate their average concentrations in indoor air when the spray product is used. We selected glycol and glycol ethers as the main target compounds, as these chemicals were detected at high frequencies and concentrations in a national survey of Japanese indoor air pollution. The extraction of these chemicals using graphite carbon cartridges was examined, with good recoveries and reproducibilities being obtained. Eighteen chemicals were analyzed in 54 commercial products and 8 chemicals were detected. More specifically, dipropylene glycol (DPG) was present in 44 samples (1.1 × 101-1.8 × 104 µg/mL); propylene glycol (PG) was present in 22 samples (1.5 × 101-2.9 × 104 µg/mL); diethylene glycol monoethyl ether (DGMEE) was found in 15 samples (trace amount-1.9 × 103 µg/mL); diethylene glycol (DEG) was present in 9 samples (1.0 × 101-2.4 × 103 µg/mL); 1,3-butandiol (13BG) was found in 5 samples (trace amount-7.4 × 103 µg/mL); 2-ethyl-1-hexanol (2E1H) was detected in 5 samples (3.2 × 10-1-4.4 × 101 µg/mL); diethylene glycol monobutyl ether (DGMBE) was present in 4 samples (2.1 × 101-7.1 × 101 µg/mL); and 3-methoxy-3-methylbutanol (MMB) was found in 2 samples (2.4 × 101-4.7 × 102 µg/mL). In addition, the average concentrations of these chemicals in indoor air were estimated using their maximum concentrations observed in the spray product. The estimated average concentrations of the chemicals in indoor air were determined to range between 1.0 × 10-2 and 1.0 mg/m3, with the exception of 2E1H and DGMBE. Furthermore, the estimated average concentrations of PG, 13BG, and DGMEE in indoor air were comparable to or higher than those reported in a national survey of Japanese indoor air pollution. It therefore appeared that household water-based hand pump sprays may contribute to the presence of these chemicals in indoor air. In contrast, estimated average concentrations of 2E1H in indoor air were low, its concentrations observed in a national survey of Japanese indoor air pollution are likely due to the use of plasticizers and paints.

Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

Glucuronidation of tizoxanide, an active metabolite of nitazoxanide, in liver and small intestine: Species differences in humans, monkeys, dogs, rats, and mice and responsible UDP-glucuronosyltransferase isoforms in humans.

Tizoxanide (TZX) is an active metabolite of nitazoxanide (NTZ) originally developed as an antiparasitic agent, and is predominantly metabolized into TZX glucuronide. In the present study, TZX glucuronidation by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice, and recombinant human UDP-glucuronosyltransferase (UGT) were examined. The kinetics of TZX glucuronidation by the liver and intestinal microsomes followed the Michaelis-Menten or biphasic model, with species-specific variations in the intrinsic clearance (CLint). Rats and mice exhibited the highest CLint values for liver microsomes, while mice and rats were the highest for intestinal microsomes. Among human UGTs, UGT1A1 and UGT1A8 demonstrated significant glucuronidation activity. Estradiol and emodin inhibited TZX glucuronidation activities in the human liver and intestinal microsomes in a dose-dependent manner, with emodin showing stronger inhibition in the intestinal microsomes. These results suggest that the roles of UGT enzymes in TZX glucuronidation in the liver and small intestine differ extensively across species and that UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. This study presents the relevant and novel-appreciative report on TZX metabolism catalyzed by UGT enzymes, which may aid in the assessment of the antiparasitic, antibacterial, and antiviral activities of NTZ for the treatment of various infections.

cDNA cloning and functional analysis of minipig uridine diphosphate-glucuronosyltransferase 1A1.

Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CL value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CL value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.

Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases.

Phthalates are widely used plasticizers that are primarily and rapidly metabolized to monoester phthalates in mammals. In the present study, the hydrolysis of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in the human liver, small intestine, kidney, and lung was examined by the catalytic, kinetic, and inhibition analyses using organ microsomal and cytosolic fractions and recombinant carboxylesterases (CESs). The Vmax (y-intercept) values based on the Eadie-Hofstee plots of DBP hydrolysis were liver > small intestine > kidney > lung in microsomes, and liver > small intestine > lung > kidney in cytosol, respectively. The CLint values (x-intercept) were small intestine > liver > kidney > lung in both microsomes and cytosol. The Vmax and CLint or CLmax values of DEHP hydrolysis were small intestine > liver > kidney > lung in both microsomes and cytosol. Bis(4-nitrophenyl) phosphate (BNPP) effectively inhibited the activities of DBP and DEHP hydrolysis in the microsomes and cytosol of liver, small intestine, kidney, and lung. Although physostigmine also potently inhibited DBP and DEHP hydrolysis activities in both the microsomes and cytosol of the small intestine and kidney, the inhibitory effects in the liver and lung were weak. In recombinant CESs, the Vmax values of DBP hydrolysis were CES1 (CES1b, CES1c) > CES2, whereas the CLmax values were CES2 > CES1 (CES1b, CES1c). On the other hand, the Vmax and CLmax values of DEHP hydrolysis were CES2 > CES1 (CES1b, CES1c). These results suggest an extensive organ-dependence of DBP and DEHP hydrolysis due to CES expression, and that CESs are responsible for the metabolic activation of phthalates.

Species differences in activation of TRPA1 by resin additive-related chemicals relevant to indoor air quality.

Transient Receptor Potential Ankyrin 1 (TRPA1), which is expressed in the airways, has causative and exacerbating roles in respiratory diseases. TRPA1 is known as a target of sick building syndrome-related air pollutants, such as formaldehyde. Thus, an in vitro TRPA1 activation assay would be useful for predicting the potential risk of air pollution. In this study, we used human TRPA1 (hTRPA1)- and mouse TRPA1 (mTRPA1)-expressing cell lines to measure TRPA1 activation by the emerging indoor air pollutants 2-ethyl-1-hexanol (2-EH), a mixture of 2,2,4-trimethyl-1,3-pentanediol 1- and 3-monoisobutyrate (Texanol), and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). The results indicated that 2-EH activated both hTRPA1 and mTRPA1 in a concentration-dependent manner, whereas TXIB did not activate hTRPA1 or mTRPA1. Texanol also activated hTRPA1 in a concentration-dependent manner. In contrast, a bell-shaped concentration-dependent curve was observed for mouse TRPA1 activation by Texanol, indicating inhibitory effects at a higher concentration range, which was also reported for menthol, a typical TRPA1 modulator. To further elucidate the mechanism underlying the species difference in TRPA1 activation by Texanol, V875G and G878V mutations were introduced into hTRPA1 and mTRPA1, respectively, which were reported to be key mutations for the inhibitory effect of menthol. These mutations switched the inhibitory effects of Texanol; thus, hTRPA1/V875G, but not mTRPA1/G878V, was inhibited at higher concentrations of Texanol. These results indicate that Texanol shares an interaction site with menthol. Overall, these findings suggest that careful interpretation is necessary when extrapolating rodent TRPA1-dependent toxicological effects to humans, especially with respect to the risk assessment of indoor air pollutants.

Initial response and cellular protection through the Keap1/Nrf2 system during the exposure of primary mouse hepatocytes to 1,2-naphthoquinone.

Quinones are reactive chemical species that cause cellular damage by modifying protein thiols and/or catalyzing the reduction of oxygen to reactive oxygen species, thereby promoting oxidative stress. Transcription factor Nrf2 plays a crucial role in cellular defense against electrophilic modification and oxidative stress. In studies using 1,2-naphthoquinone (1,2-NQ) as a model quinone, we found that Keap1, the negative regulator of Nrf2, was readily arylated at its reactive thiols by 1,2-NQ. Exposure of primary mouse hepatocytes to 1,2-NQ resulted in the activation of Nrf2 and the upregulation of some of Nrf2's downstream genes. This interaction was further investigated in hepatocytes from Nrf2 knockout mice in which the proteins responsible for the metabolism and excretion of 1,2-NQ are minimally expressed. The chemical modification of cellular proteins by 1,2-NQ was enhanced by Nrf2 deletion, resulting in increased toxicity. However, deletion of the negative regulatory protein, Keap1, drastically reduced the covalent binding by 1,2-NQ and its cellular toxicity. Experiments with chemicals that inhibit the biotransformation and extracellular excretion of 1,2-NQ suggest that 1,2-NQ undergoes detoxification and excretion into the extracellular space predominantly by two-electron reduction and subsequent glucuronidation by NAD(P)H:quinone oxidoreductase 1 and uridine 5'-diphosphate-glucuronosyltransferases, followed by multidrug resistance-associated protein-dependent excretion. These findings suggest that the Keap1/Nrf2 system is essential for the prevention of cell damage resulting from exposure to 1,2-NQ.

[Volatile organic compounds (VOCs) emitted from large furniture].

Indoor air pollution by volatile organic compounds (VOCs), which may cause a hazardous influence on human being such as sick building (sick house) syndrome, has become a serious problem. In this study, VOCs emitted from nine pieces of home furniture, three sets of dining tables, three sets of chest of drawers and three sofas, were analyzed as potential sources of indoor air pollution by large chamber test method (JIS A 1911). Based on the emission rates of total VOC (TVOC), the impacts on the indoor TVOC was estimated by the sample model with a volume of 20 m3 and ventilation frequency of 0.5 times/h. The estimated TVOC increment values were exceeded the provisional target value for indoor air (400 microg/m3) in three sets of dining tables, one set of chest of drawer and one sofa. The estimated increment of formaldehyde were exceeded the guideline value (100 microg/m3) in one set of dining table, two sets of chest of drawers and one sofa. These results revealed that VOC emissions from furniture may influence significantly indoor air quality. Also, in this study, to establish the alternative method for large chamber test methods, emission rates from representative three parts of furniture unit were evaluated using the small chamber and emission rate from full-sized furniture was predicted. Emission rates of TVOC and formaldehyde predicted by small chamber test were 3-46% and 6-252% of the data obtained using large chamber test, respectively.

[Volatile organic compounds (VOCs) emitted from wood furniture--estimation of emission rate by passive flux sampler].

The aim of this study was to evaluate aldehydes and other volatile organic compounds (VOCs) emission from furniture, which may cause hazardous influence on human being such as sick building/sick house syndrome. In this study, VOCs emitted from six kinds of wood furniture, including three set of dining tables and three beds, were analyzed by large chamber test method (JIS A 1911). Based on the emission rates of total VOCs (TVOC), the impacts on the indoor TVOC was estimated by the simulation model with volume of 20 m3 and ventilation frequency of 0.5 times/h. The estimated increment of formaldehyde were exceeded the guideline value (100 microg/m3) in one set of dining table and one bed. The estimated TVOC increment values were exceeded the provisional target value for indoor air (400 microg/m3) in two sets of dining tables and two beds. These results revealed that VOC emissions from wood furniture may influence significantly indoor air quality. Also, in this study, to establish the alternative method for large chamber test methods, emission rates from representative five areas of furniture unit were evaluated by passive sampling method using flux sampler and emission rate from full-sized furniture was predicted. Emission rates predicted by flux passive sampler were 10-106% (formaldehyde) and 8-141% (TVOC) of the data measured using large chamber test, respectively.