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.

Predictability of plasma concentration-time curves in humans using single-species allometric scaling of chimeric mice with humanized liver.

1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration-time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration-time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration-time curve in drug discovery, although there are some limitations.

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

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

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.

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

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. 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. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. 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. 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. 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.

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.

Predictability of metabolism of ibuprofen and naproxen using chimeric mice with human hepatocytes.

Prediction of human drug metabolism is important for drug development. Recently, the number of new drug candidates metabolized by not only cytochrome P450 (P450) but also non-P450 has been increasing. It is necessary to consider species differences in drug metabolism between humans and experimental animals. We examined species differences of drug metabolism, especially between humans and rats, for ibuprofen and (S)-naproxen as nonsteroidal anti-inflammatory drugs, which are metabolized by P450 and UDP-glucuronosyltransferase, sulfotransferase, and amino acid N-acyltransferase for taurine conjugation in liver, using human chimeric mice (h-PXB mice) repopulated with human hepatocytes and rat chimeric mice (r-PXB mice) transplanted with rat hepatocytes. We performed the direct comparison of excretory metabolites in urine between h-PXB mice and reported data for humans as well as between r-PXB mice and rats after administration of ibuprofen and (S)-naproxen. Good agreement for urinary metabolites (percentage of dose) was observed not only between humans and h-PXB mice but also between rats and r-PXB mice. Therefore, the metabolic profiles in humans and rats reflected those in h-PXB mice and r-PXB mice. Our results indicated that h-PXB mice should be helpful for predicting the quantitative metabolic profiles of drugs mediated by P450 and non-P450 in liver, and r-PXB mice should be helpful for evaluation of species differences in these metabolic enzymes.

Prediction of in vivo hepatic clearance and half-life of drug candidates in human using chimeric mice with humanized liver.

Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. Direct comparison between in vitro intrinsic clearance (CL(int,in vitro)) in PXB mice hepatocytes and in vivo intrinsic clearance (CL(int,in vivo)) in humans, calculated based on a well stirred model, showed a moderate correlation (r² = 0.475, p = 0.009). However, when CL(int,in vivo) values in humans and PXB mice were compared similarly, there was a good correlation (r² = 0.754, p = 1.174 × 10⁻4). Elimination half-life (t(1/2)) after intravenous administration also showed a good correlation (r² = 0.886, p = 1.506 × 10⁻4) between humans and PXB mice. The rank order of CL and t(1/2) in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions.

Xanthine oxidase and aldehyde oxidase contribute to allopurinol metabolism in rats.

BACKGROUND: Allopurinol is used to treat hyperuricemia and gout. It is metabolized to oxypurinol by xanthine oxidase (XO), and aldehyde oxidase (AO). Allopurinol and oxypurinol are potent XO inhibitors that reduce the plasma uric acid levels. Although oxypurinol levels show large inter-individual variations, high concentrations of oxypurinol can cause various adverse effects. Therefore, it is important to understand allopurinol metabolism by XO and AO. In this study we aimed to estimate the role of AO and XO in allopurinol metabolism by pre-administering Crl:CD and Jcl:SD rats, which have known strain differences in AO activity, with XO inhibitor febuxostat. METHODS: Allopurinol (30 or 100 mg/kg) was administered to Crl:CD and Jcl:SD rats with low and high AO activity, respectively, after pretreatment with or without febuxostat. The serum concentrations of allopurinol and oxypurinol were measured, and the area under the concentration-time curve (AUC) was calculated from the 48 h serum concentration-time profile. In vivo metabolic activity was measured as the ratio AUCoxypurinol /AUCallopurinol. RESULTS: Although no strain-specific differences were observed in the AUCoxypurinol/AUCallopurinol ratio in the allopurinol (30 mg/kg)-treated group, the ratio in Jcl:SD rats was higher than that in Crl:CD rats after febuxostat pretreatment. Contrastingly, the AUC ratio of allopurinol (100 mg/kg) was approximately 2-fold higher in Jcl:SD rats than that in Crl:CD rats. These findings showed that Jcl:SD rats had higher intrinsic AO activity than Crl:CD rats did. However, febuxostat pretreatment substantially decreased the activity, as measured by the AUC ratio using allopurinol (100 mg/kg), to 46 and 63% in Crl:CD rats and Jcl:SD rats, respectively, compared to the control group without febuxostat pretreatment. CONCLUSIONS: We elucidated the role of XO and AO in allopurinol metabolism in Crl:CD and Jcl:SD rats. Notably, AO can exert a proportionately greater impact on allopurinol metabolism at high allopurinol concentrations. AO's impact on allopurinol metabolism is meaningful enough that individual differences in AO may explain allopurinol toxicity events. Considering the inter-individual differences in AO activity, these findings can aid to dose adjustment of allopurinol to avoid potential adverse effects.

Inhibitory effects of organophosphate esters on carboxylesterase activity of rat liver microsomes.

We investigated the inhibitory effects of 13 organophosphate esters (OPEs) and hydrolytic metabolites on the carboxylesterase activity of rat liver microsomes in vitro in order to examine whether there might be a potential impact on human health, and to elucidate the structure activity relationship. Among the test compounds, 2-ethylhexyl diphenyl phosphate (EDPhP) was the most potent inhibitor of carboxylesterase activity, as measured in terms of 4-nitrophenol acetate hydrolase activity, followed by tri-m-cresyl phosphate (TmCP), cresyl diphenyl phosphate (CDPhP) and triphenyl phosphate (TPhP). The IC50 values were as follows: EDPhP (IC50: 0.03 µM) > TmCP (0.4 µM) > CDPhP (0.8 µM) > TPhP (14 µM) > tris(1,3-dichloro-2-propyl) phosphate (17 µM) > tris(2-ethylhexyl) phosphate (77 µM) > tri-n-propyl phosphate (84 µM) > tris(2-chloroethyl) phosphate (104 µM) > tris(2-butoxyethyl) phosphate (124 µM) > tri-n-butyl phosphate (230 µM). The IC50 value of EDPhP was three orders of magnitude lower than that of bis(4-nitrophenyl) phosphate, which is widely used as an inhibitor of carboxylesterase. Trimethyl phosphate, triethyl phosphate and tris(2-chloroisopropyl) phosphate slightly inhibited the carboxylesterase activity; their IC50 values were above 300 µM. Lineweaver-Burk plots indicated that the inhibition by several OPEs was non-competitive. Diphenyl and monophenyl phosphates, which are metabolites of TPhP, showed weaker inhibitory effects than that of TPhP.

Amiodarone bioconcentration and suppression of metamorphosis in Xenopus.

Trace concentrations of a number of pharmaceutically active compounds have been detected in the aquatic environment in many countries, where they are thought to have the potential to exert adverse effects on non-target organisms. Amiodarone (AMD) is one such high-risk compound commonly used in general hospitals. AMD is known to alter normal thyroid hormone (TH) function, although little information is available regarding the specific mechanism by which this disruption occurs. Anuran tadpole metamorphosis is a TH-controlled developmental process and has proven to be useful as a screening tool for environmental pollutants suspected of disrupting TH functions. In the present study, our objective was to clarify the effects of AMD on Xenopus metamorphosis as well as to assess the bioconcentration of this pharmaceutical in the liver. We found that AMD suppressed spontaneous metamorphosis, including tail regression and hindlimb elongation in pro-metamorphic stage tadpoles, which is controlled by endogenous circulating TH, indicating that AMD is a TH antagonist. In transgenic X. laevis tadpoles carrying plasmid DNA containing TH-responsive element (TRE) and a 5'-upstream promoter region of the TH receptor (TR) ßA1 gene linked to a green fluorescent protein (EGFP) gene, triiodothyronine (T3) exposure induced a strong EGFP expression in the hind limbs, whereas the addition of AMD to T3 suppressed EGFP expression, suggesting that this drug interferes with the binding of T3 to TR, leading to the inhibition of TR-mediated gene expression. We also found AMD to be highly bioconcentrated in the liver of pro-metamorphic X. tropicalis tadpoles, and we monitored hepatic accumulation of this drug using mass spectrometry imaging (MSI). Our findings suggest that AMD imposes potential risk to aquatic wildlife by disrupting TH homeostasis, with further possibility of accumulating in organisms higher up in the food chain.

Current situation of drug information in the kindergarten and nursery teacher: a pilot study.

Because children cannot be expected to take medications correctly by themselves, parents are responsible for administering drugs based on the information provided by pharmacists. It has been reported that 90% of children aged 3-5 years in Japan attend kindergarten or nursery school, where teachers are responsible for the administration of some drugs to children. This study evaluated the types of information that teachers receive from parents. We conducted a questionnaire-based survey on drug information imparted to 144 teachers working in kindergarten or nursery schools in Hiroshima and Kure. The teachers reported that drug information from parents mainly comprised dosage and usage. However, little information was provided concerning the drug name, adverse drug reactions, and interaction with food items. To administer drugs to children safely, kindergarten and nursery teachers considered the information regarding adverse drug reactions (111/123 teachers), interaction with foods (106/123 teachers), and effective means of administering drugs (117/123 teachers) as important. The pharmacists' prescription notes have information on dosage, usage, drug name, adverse drug reactions, and interaction with food items. However, the teachers receive drug information from parents in the order of oral communication, a written note, and via the pharmacists' prescription note. Seventy-two percent of teachers (89/123 teachers) insisted on needing the pharmacists' prescription note. These results suggest that teachers are uncomfortable administering medications to children primarily due to inadequate information. Pharmacists should instruct parents to provide teachers with prescription notes to prevent grave medication errors.

Profiling of bisphenol A and eight its analogues on transcriptional activity via human nuclear receptors.

Several bisphenol A (BPA) analogues have been detected in environmental samples, foodstuffs, and/or human biological samples, and there is concern regarding their potential endocrine-disrupting effects. In this study, we characterized the agonistic and/or antagonistic activities of BPA and eight its analogues against human estrogen receptors (ERα/ß), androgen receptor (AR), glucocorticoid receptor (GR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR). All the test compounds, except for bisphenol P (BPP), showed both ERα and ERß agonistic activities, with bisphenol AF (BPAF) being the most potent. On the other hand, BPAF and BPP showed ERα and ERß antagonistic activities. Interestingly, their ER activities demonstrated a preference toward ERß. All the test compounds, except for bisphenol S, showed AR antagonistic activities, with bisphenol E being the most potent. Weak GR antagonistic activities were also found in BPA and five its analogues. PXR agonistic activity was observed in the six compounds, with bisphenol Z being the most potent. Results of the CAR assay revealed that BPA and five its analogues acted as CAR inverse agonists. Taken together, these results suggested that BPA analogues demonstrate multiple effects via human nuclear receptors in a similar manner to BPA, and several analogues might have more potent endocrine-disrupting activity than does BPA.

CYP2C9-catalyzed metabolism of S-warfarin to 7-hydroxywarfarin in vivo and in vitro in chimeric mice with humanized liver.

Chimeric mice having humanized livers were constructed by transplantation of human hepatocytes. In this study, we investigated whether these mice have a capacity for drug metabolism similar to that of humans by examining hydroxylation of S-warfarin, which is predominantly metabolized to S-7-hydroxywarfarin, catalyzed by CYP2C9, in humans but not mice. The 7-hydroxylating activity of chimeric mouse liver microsomes toward S-warfarin was approximately 10-fold higher than that of control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice. The 7-hydroxylase activity of chimeric mouse liver microsomes was markedly inhibited by sulfaphenazole, as was that of human liver microsomes, whereas the activity of control mice was unaffected. The CYP2C isoform in chimeric mouse liver was also confirmed to be the human isoform, CYP2C9, by immunoblot analysis. In the present in vivo study, the level of S-7-hydroxywarfarin in plasma of chimeric mice was approximately 7-fold higher than that in control mice, in agreement with the in vitro data. Thus, the CYP2C isoform in chimeric mice functions in vivo and in vitro as a human isoform, CYP2C9. These results suggest that chimeric mice with humanized liver could be useful for predicting drug metabolism in humans, at least regarding CYP2C9-dependent metabolism.

Aldehyde oxidase-catalyzed metabolism of N1-methylnicotinamide in vivo and in vitro in chimeric mice with humanized liver.

Aldehyde oxidase-mediated oxidation of N(1)-methylnicotinamide to N(1)-methyl-2-pyridine-5-carboxamide (2-PY) and N(1)-methyl-4-pyridone-5-carboxamide (4-PY) in chimeric mice constructed by transplanting human hepatocytes into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice was examined in vivo and in vitro. The activity in liver cytosol of chimeric mice with a high replacement index was approximately 4-fold higher than that in control mice. Furthermore, the oxidation products in control mice were 2-PY and 4-PY, whereas, in chimeric mice, the major product was 2-PY, as in humans. The aldehyde oxidase in chimeric mouse liver was confirmed to be of human type by immunoblotting analysis. The ratio of pyridones (2-PY/4-PY) excreted in the urine of chimeric mice was closer to that of humans than to that of control mice. Thus, the aldehyde oxidase in chimeric mice has human-type functional characteristics.

[Effect of Environmental Factors on the Ecotoxicity of Pharmaceuticals and Personal Care Products].

　In recent years, pharmaceuticals and personal care products (PPCPs) have emerged as significant pollutants of aquatic environments and have been detected at levels in the range of ng/L to µg/L. The source of PPCPs is humans and livestock that have been administered pharmaceuticals and subsequently excreted them via urine and feces. Unlike agricultural chemicals, the environmental dynamics of PPCPs is not examined and they would undergo structural transformation by environmental factors, e.g., sunlight, microorganisms and treatments in sewage treatment plants (STPs). Processing at STPs can remove various PPCPs; however, they are not removed completely and some persist in the effluents. In this study, we examined the degradation of 9 pharmaceuticals (acetaminophen, amiodarone, dapsone, dexamethasone, indomethacin, raloxifene, phenytoin, naproxen, and sulindac) by sunlight or UV, and investigated the ecotoxicological variation of degradation products. Sunlight (UVA and UVB) degraded most pharmaceuticals, except acetaminophen and phenytoin. Similar results were obtained with UVB and UVA. All the pharmaceuticals were photodegraded by UVC, which is used for sterilization in STPs. Ecotoxicity assay using the luminescent bacteria test (ISO11348) indicated that UVC irradiation increased the toxicity of acetaminophen and phenytoin significantly. The photodegraded product of acetaminophen was identified as 1-(2-amino-5-hydroxyphenyl)ethanone and that of phenytoin as benzophenone, and the authentic compounds showed high toxicity. Photodegraded products of PPCPs are a concern in ecotoxicology.

Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17ß-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.

Developmental changes of aldehyde oxidase in postnatal rat liver.

In this study, the developmental changes and variability of aldehyde oxidase in postnatal rat liver were examined. Postnatal day 1, 7 and 14 rats showed little or no liver aldehyde oxidase activity, as evaluated in terms of the activities for oxidation of benzaldehyde to benzoic acid, N-1-methylnicotinamide (NMN) to N-1-methyl-2-pyridone-5-carboxamide (2-PY) and N-1-methyl-4-pyridone-3-carboxamide (4-PY), and methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX). However, these oxidase activities were markedly increased in liver cytosol from the rats after postnatal day 14. The activity was then maintained up to 6 weeks. The amounts of 2-PY and 4-PY formed from NMN were almost the same. The development of aldehyde oxidase activity toward benzaldehyde was closely correlated with that of oxidase activity toward NMN and MTX. The expression of aldehyde oxidase at postnatal day 14 was confirmed by Western blotting analysis. The density of bands of aldehyde oxidase was closely correlated with the oxidase activity toward benzaldehyde. The developmental changes of aldehyde oxidase activities during postnatal reflected the changes in the amount of the oxidase protein. Thus, aldehyde oxidase activity in rats rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated by expression of the protein.

The in vitro metabolism of a pyrethroid insecticide, permethrin, and its hydrolysis products in rats.

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.