Metabolism of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and its isomer, N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), in rats. 1. Identification of metabolites in feces and urine.

Rats were orally dosed with a 1:1 diastereomixture of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), both labeled with 14C, at 200 mg/kg/day for 5 consecutive days, and 16 metabolites in urine and feces were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Several of them were unique decyanated and/or cyclic compounds (lactone, imide, cyclic amide, cyclic imino ether forms). Major biotransformation reactions of the mixture of S-2900 and S-2900S in rats are proposed on the basis of the metabolites identified in this study.

Simulation of the toxicokinetics of trichloroethylene, methylene chloride, styrene and n-hexane by a toxicokinetics/toxicodynamics model using experimental data.

The toxicokinetics/toxicodynamics (TKTD) model simulates the toxicokinetics of a chemical based on physiological data such as blood flow, tissue partition coefficients and metabolism. In this study, Andersen and Clewell's TKTD model was used with seven compartments and ten differential equations for calculating chemical balances in the compartments (Andersen and Clewell 1996, Workshop on physiologically-based pharmacokinetic/pharmacodynamic modeling and risk assessment, Aug. 5-16 at Colorado State University, U.S.A) . Using this model, the authors attempted to simulate the behavior of four chemicals: trichloroethylene, methylene chloride, styrene and n-hexane, and the results were evaluated. Simulations of the behavior of trichloroethylene taken in via inhalation and oral exposure routes were also done. The differences between simulations and measurements are due to the differences between the absorption rates of the exposure routes. By changing the absorption rates, the simulation showed agreement with the measured values. The simulations of the other three chemicals showed good results. Thus, this model is useful for simulating the behavior of chemicals for preliminary toxicity assessment.

Pharmacokinetics of a novel N-methyl-D-aspartate receptor antagonist (SM-18400): identification of an N-acetylated metabolite and pre-clinical assessment of N-acetylation polymorphism.

(S)-9-chloro-5-[p-aminomethyl-o-(carboxymethoxy)phenylcarbamoylmethyl]-6,7-dihydro-1 H,5 H-pyrido[1,2,3-de]quinoxaline-2,3-dione hydrochloride trihydrate (SM-18400) was given intravenously to rats and dogs and its pharmacokinetics was investigated. By LC/MS/MS analysis, the major metabolite in the rat serum was identified as N-acetylated SM-18400 (SM-NAc). In rats, AUC ratio of SM-NAc to SM-18400 was approximately 50%. However, 71% of the dose was excreted as unchanged SM-18400 and only 9.8% as SM-NAc in the urine and bile, indicating that the contribution of N-acetylation clearance (CL(NAc)) to the total clearance (CL(tot)) is limited to 10-30% in rats. No SM-NAc or other metabolites were detected in the dog serum, urine or bile. The in vitro intrinsic clearance (CL(int), ml/min/mg cytosolic protein) of N-acetyltransferase (NAT) activities of dog liver cytosol towards SM-18400 and hepatic N-acetylation clearance (CL(NAc), ml/min/kg body weight) estimated by well-stirred model were both only 5% of the respective rat value, well reflecting the relative in vivo CL(NAc)/CL(tot) ratios. CL(int) values for human liver cytosol samples (n = 4) and estimated CL(NAc) were all less than 18% and 7% of the rat, respectively. Based on these results, we concluded that the CL(NAc)/CL(tot) of human would be small enough to avoid major inter-individual variance in SM-18400 pharmacokinetics due to N-acetylation polymorphism. In addition, even a human liver cytosol sample lacking polymorphic NAT2 activity as determined by sulfamethazine (SMZ) N-acetylation analysis, proved capable of acetylating SM-18400, suggesting that NAT2 is not the major enzyme responsible for N-acetylation of SM-18400 in human. This fact would also reduce the risk of N-acetylation polymorphism playing a role in clinical use of this drug.

In vitro metabolism of perospirone in rat, monkey and human liver microsomes.

In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using 14C labeled perospirone. With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain. However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent. Unknown metabolites accounted for about 10% of the total. After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring. After incubation for 60 minutes, ID-15001, i.e. the butylene chain cleavage type increased. Unknown metabolites accounted for about 20%. After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring. In addition, MX11 and many unknown metabolites were evident. After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased. Individual differences were found in the metabolic reaction rate. With human liver microsomes. MX11, ID-15001 and unknown metabolites were again the major metabolites. With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly. Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug. The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation. The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced.

Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3.

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-gamma on alpha2(I) procollagen gene (COL1A2) transcription through the IFN-gamma response element located between -161 and -150. Here we report that YB-1 counter-represses TGF-beta-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around -265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-gamma-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-gamma in the absence and presence of TGF-beta. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-beta and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-gamma/YB-1 counteracts TGF-beta/Smad3. They also indicate that IFN-gamma/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-gamma response element and through a cross-talk with the TGF-beta/Smad signaling pathway.

Y-box-binding protein YB-1 mediates transcriptional repression of human alpha 2(I) collagen gene expression by interferon-gamma.

We have demonstrated previously that a proximal element within the human alpha2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-gamma (IFN-gamma), and designated this region the IFN-gamma response element (IgRE). Screening of a human fibroblast cDNA expression library with a radiolabeled IgRE probe exclusively yielded clones with a sequence identical to that of the transcription factor YB-1. Electrophoretic mobility shift assays (EMSA) using various IgRE-derived oligonucleotide probes containing serial two-base mutations showed that YB-1 protein was preferentially bound to the pyrimidine-rich sequence within the IgRE. This region is located immediately downstream of and partly overlaps the previously reported Sp1/Sp3 binding site. Overexpression of YB-1 in human dermal fibroblasts decreased steady state levels of COL1A2 mRNA and repressed COL1A2 promoter activity in a dose-dependent manner. This inhibitory effect of YB-1 on COL1A2 expression was abolished by mutations of the IgRE shown to prevent YB-1 binding in EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-gamma, suggesting that YB-1 mediates the inhibitory action of IFN-gamma on COL1A2 promoter through its binding to the IgRE. Also, overexpression of a deletion mutant YB-1, which lacks the carboxyl-terminal domain, abrogated the repression of COL1A2 transcription by IFN-gamma. A functional correlation between IFN-gamma and YB-1 was further supported by luciferase assays using four tandem repeats of the Y-box consensus oligonucleotide linked to a minimal promoter. EMSA and Western blot analysis using cytoplasmic and nuclear proteins implied that IFN-gamma promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-gamma was further provided by using a YB-1-green fluorescent protein expression plasmid transfected into human fibroblasts. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-gamma-elicited inhibition of COL1A2 expression, namely YB-1.

Lack of changes in brain muscarinic receptor and motor activity of mice after neonatal inhalation exposure to d-allethrin.

Synthetic pyrethroids are among the most common pesticides and insecticides currently in worldwide use. Eriksson and co-workers postulated that oral exposure of mice to pyrethroids during a neonatal brain growth spurt induces permanent disturbance in the cerebral muscarinic cholinergic receptor (MAChR) and behaviour. However, the scientific basis for these phenomena is now under discussion. The present study was performed to determine whether the experimental findings of Eriksson's study could be reproduced in newborn mice by inhalation. Male and female NMRI mice were exposed to d-allethrin by whole-body inhalation for 6 h per day between postnatal days 10 and 16. Actual concentrations of d-allethrin were 0.43, 1.35, 3.49 and 74.2 mg m(-3) (equivalent to 0.70, 2.2, 5.7 and 120.2 mg kg(-1) day(-1), respectively), and the mass median aerodynamic diameter and geometric log-standard deviation of mist particles ranged from 2.58 to 2.98 micro m and from 1.58 to 2.09 micro m for all groups, respectively. The highest exposure level in the present study (74.2 mg m(-3)) was ca. 13,000 times as high as the concentration used in practice. The MAChR in the three brain areas (cortex, hippocampus and striatum) and motor activity were examined at the ages of 17 days and 4 months. In addition, a water-maze test was performed at the age of 11 months. There was no systemic toxicity interfering with the interpretation of assay results. The neonatal exposure to d-allethrin by inhalation did not induce effects either on the brain MAChR density and motor activity at 17 days and 4 months or on performance in the learning/memory test at the age of 11 months. The effects of allethrins on developmental neurotoxicity that Eriksson and co-workers reported previously were not reproduced in the present study.

Lack of (anti-) androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays.

Synthetic pyrethroids are among the most common pesticides and insecticides currently in use worldwide. Recently, chemicals classified as synthetic pyrethroids are suspected as being endocrine disrupting chemicals. However, no study has been conducted to assess their potential hormonal activities using in vivo test specifically focused on endocrine disruption. In the present study, we evaluated the interaction of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) with androgen receptor (AR)- and estrogen receptor (ER)-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the Hershberger and uterotrophic assays have not yet been fully developed, both are widely used and are being considered by OECD as short-term screening assays for hormonal activity. A 5-day Hershberger assay using castrated male rats measures agonistic and androgenic ability of the test chemicals to AR of several accessory glands/tissues (the ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands, and levator ani plus bulbocavernosus muscles). Esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (25, 50, or 75 mg/kg/day) was administered by oral gavage for 5 days to castrated male Crj:CD(SD)IGS rats (1 week after the castration, 11 weeks of age) with or without coadministration of 0.25 mg/kg/day testosterone propionate (subcutaneous injection on the dorsal surface). The highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. None of esfenvalerate, fenvalerate, and permethrin showed any androgenic or antiandrogenic effects. Reference control of p,p'-DDE and methyltestosterone (100 mg/kg/day) provided significant effects in this assay protocol. Potential effects of these pyrethroids mediated through the ER were evaluated by means of 3-day uterotrophic assay using ovariectomized Crj:CD(SD)IGS rats (2 weeks after the ovariectomy, 8 weeks of age). No increase in weight of uterus (wet or blotted) was observed following oral exposure to esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (37.5, 75, or 150 mg/kg/day), respectively. Again, the highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. Reference controls consisting of ethynyl estradiol (0.03 mg/kg/day) and methoxychlor (125 mg/kg/day) both showed a significant effect in this assay protocol. It is concluded that, based on the results of these two reliable in vivo assays, none of esfenvalerate, fenvalerate, or permethrin exhibit any potential to cause adverse (anti-) androgenic or estrogenic effects at dose levels below that of those causing excessive systemic toxicity.