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.
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.
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.
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.
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. CL
int
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. CL
int
values were rats > monkeys > mice > dogs > humans. The tissue dependence of CL
int
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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. K
m
, V
max
, and CL
int
values in human liver microsomes were 7.54 µM, 17.7 nmol/min/mg protein, and 2.36 mL/min/mg protein, respectively. CL
int
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, K
m
, V
max
, and CL
int
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 CL
int
in monkey, dogs, rats, and mice to that of humans were 7.0-, 12-, 34-, and 29-fold, respectively. Although CL
int
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Naringenin, a flavanone found in citrus fruits, is mainly metabolized into glucuronide(s) by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the glucuronidation of ...naringenin in the liver and intestine microsomes of humans, monkeys, rats, and mice was examined. The kinetics of 7-glucuronidation in human liver and intestine microsomes followed the Michaelis-Menten model. Kinetics in mouse liver and intestine microsomes also followed the Michaelis-Menten model, whereas those in monkey and rat liver microsomes fit the biphasic model. Kinetics in monkey and rat intestine microsomes fit the Michaelis-Menten and substrate inhibition models, respectively. CLint values were mice > monkeys > rats > humans for liver microsomes, and mice > rats > monkeys > humans for intestine microsomes. In 4´-glucuronidation, activities in human liver microsomes and monkey liver and intestine microsomes were negligible or very low. Kinetics in rat and mouse liver microsomes followed the biphasic and Michaelis-Menten models, respectively. CLint values were rats > mice for liver microsomes, and rats > mice > humans for intestine microsomes. These results suggest that the metabolic abilities and regioselectivity of UGT enzymes toward naringenin in the liver and intestines generally differ between primates and rodents.
•Naringenin regioselective glucuronidation in mammals were examined in vitro system.•Hepatic 7-glucuronidation activities were mice > monkeys > rats > humans.•Intestinal 7-glucuronidation activities were mice > rats ≥ monkeys > humans.•Heapatic 4′-glucuronidation activities were mice > rats > humans >> monkeys.•Intestinal 4′-glucuronidation activities were mice > rats >> humans and monkeys.
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.
Favipiravir is an anti‐viral agent effective against several RNA viruses that is converted into an inactive oxidative metabolite (M1), mainly by aldehyde oxidase, in humans. The activities in male and female humans were 297 and 241 pmol/min/mg protein, respectively. The activities in monkeys were 8.4‐fold for males and 12‐fold for females higher than those in humans, respectively. The activities in male and female rats were markedly lower than those in humans, with relative levels of 8.1% and 12%, respectively. In mice, the activity levels compared with humans were 2.0‐fold for males and 0.48‐fold for females, and a sex difference (male > female) of approximately 5‐fold was observed. This suggests that the roles of aldehyde oxidase in the hepatic metabolism of favipiravir extensively differ depending on species and sex, and this study will aid in the assessment of the anti‐viral activities of favipiravir against novel and/or variant viruses.
Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that is rapidly metabolized to mono(2-ethylhexyl) phthalate (MEHP), an active metabolite, in mammals. In the present study, the ...hydrolysis of DEHP by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice was examined. The kinetics of liver microsomes fit the Michaelis-Menten model for humans, monkeys, and rats, and the Hill model for dogs and mice. Km or S50 values were similar among species, whereas Vmax exhibited species differences of approximately 9-fold. CLint or CLmax values were in the order of mice > dogs > monkeys ≥ rats > humans. Hydrolytic activity towards DEHP was not detected in the intestinal microsomes of humans or dogs. The kinetics of monkeys, rats, and mice followed the Hill model. In comparisons of the liver microsomes of each species, S50 values were similar, while Vmax and CLmax values (mice > rats > monkeys) were considerably lower (approximately 5–25%). These results suggest that hydrolytic activity towards DEHP in the liver and intestines markedly differ among humans and non-rodent and rodent experimental animals, and imply that species differences are closely associated with the toxicity of DEHP.
•DEHP hydrolysis in humans and experimental animals was examined in an in vitro system.•Hepatic hydrolysis activities were mice > dogs > monkeys ≥ rats > humans.•Intestinal hydrolysis activities were not detected in humans and dogs.•Intestinal hydrolysis activities were mice > rats > monkeys.
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 CL
int
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 CL
int
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.
