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The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancer-dependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)-driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation.

Background Pregnane X receptor (PXR) is a key regulator of the induction of drug metabolizing enzymes. PXR has been studied for its importance in drug-drug or herb-drug interactions, and it is also a molecular target for the treatment of inflammatory and metabolic diseases. Purpose This study aims to determine new natural PXR-ligands from traditional plant medicines. Methods The PXR activation activity was measured by a mammalian one hybrid assay of PXR. Identification of the active compound from Alisma rhizome (the rhizomes of Alisma orientale) was carried out by bioassay-guided fractionation method. The transcriptional activity of the liver-enriched nuclear receptors was measured by the luciferase reporter assay. The interaction between the SRC-1 and PXR was measured by a mammalian 2-hybrid assay. The expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes were measured by quantitative RT-PCR method. Results The extract of Alisma rhizome showed the most potent activation activity by screening of a library of medicinal plant extracts. Alisol B 23-acetate (ABA) was identified to be the active compound of Alisma rhizome. ABA caused a concentration-dependent increase on the PXR-dependent transactivation of a luciferase reporter gene, but did not affect the ligand binding activity of the liver-enriched nuclear receptors, such as CAR, LXR, FXR, PPARα, PPARδ and PPARγ, emphasizing that ABA is a potent and specific agonist of PXR. With ABA treatment, the direct interaction between the ligand-binding domain of PXR and the receptor interaction domain of SRC1 was observed. ABA also induced the expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes. Conclusion Since the rhizomes of Alisma orientale are used for a wide range of ailments in traditional Chinese medicine and Japanese Kampo medicine, this study could possibly extend into the clinical usage of these medicines via the mechanism of PXR activation.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.

The constitutive androstane receptor (CAR) is a transcription factor that belongs to the nuclear receptor superfamily. CAR binds as a heterodimer with the retinoid X receptor α (RXRα) to CAR response elements (CAREs) and regulates the expression of various drug metabolizing enzymes and transporters. To identify CAR/RXRα binding sites in the human genome, we performed a modified yeast one-hybrid assay that enables rapid and efficient identification of genomic targets for DNA-binding proteins. DNA fragments were recovered from positive yeast colonies by PCR and sequenced. A motif enrichment analysis revealed that the most frequent motif was a direct repeat (DR) of RGKTCA-like core sequence spaced by 4 bp. Next, we predicted 149 putative CAR/RXRα binding sites from 414 unique clones, by searching for DRs, everted repeats (ERs) and inverted repeats (IRs) of the RGKTCA-like core motif. Based on gel mobility shift assays, the CAR/RXRα heterodimer could directly interact with the 108 predicted sequences, which included not only classical CAREs but also a wide variety of arrangements. Furthermore, we identified 17 regulatory polymorphisms on the CAR/RXRα-binding sites that may influence individual variation in the expression of CAR-regulated genes. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of CAR/RXRα het-erodimers.

It has been noticed that crosstalk between androgen receptor (AR) and mammalian target of rapamycin (mTOR) signaling pathways plays a crucial role in the proliferation of prostate cancer cells. To clarify this mechanism, we focused on DEPTOR, a naturally occurring inhibitor of mTOR. The treatment of a human AR-positive prostate cancer cell line, LNCaP, with the AR-agonist dihydrotestosterone (DHT) repressed DEPTOR mRNA expression in a time-dependent manner. This repression was abrogated by treatment with the AR-antagonist bicalutamide. Knockdown of DEPTOR mRNA by siRNA resulted in the increased phosphorylation of 70 kDa ribosomal protein S6 kinase 1 (S6K), a substrate of mTORC1, accompanied by the elevated expression of cyclin D1, a positive regulator of cell proliferation. Furthermore, the ChIP assay demonstrated that AR could bind to AR-responsible element-like region within the 4th intron of the DEPTOR gene. The amount of acetylated histone H3 (Lys9, Lys14) was reduced by the DHT treatment in this region. Taken together, these results propose that AR-dependent prostate cancer cell proliferation requires decreased DEPTOR transcription directly controlled by AR.

A chemical study of the roots of Euphorbia fischeriana resulted in the isolation of seven triterpenes (1-7), including two new compounds: (24R,S)-3β-24,31-epoxy-24-methylcycloartane (1) and (24R,S)-3β,31-dihydroxy-24-methoxy-24-methylcycloartane (2). Their structures were elucidated through extensive spectroscopic analyses. Cycloartanes 1-4 showed significant human CYP3A4 promoter activity through a series of luciferase reporter assays. Of these compounds, 3 and 4 activated the pregnane X receptor (PXR) and induced CYP3A4 mRNA expression in human primary hepatocytes. However, despite showing the most potent human CYP3A4 promoter activity via a PXR-independent pathway, 2 did not affect CYP3A4 mRNA expression in human primary hepatocytes. This difference is correlated to substitutions in C-24 and C-25 of the cycloartane structure.

The constitutive androstane receptor (CAR) is a nuclear receptor that acts as a transcription factor for a variety of genes, including genes encoding xenobiotic, steroid, and drug-metabolizing enzymes and transporters. Transactivation of a target gene by a transcription factor is generally mediated through the concerted and stepwise recruitment of various proteins termed coregulators, including coactivators and corepressors. In this study, TRIM24 (also known as transcriptional intermediary factor 1 alpha) was found to interact with the CAR. TRIM24 enhanced the CAR-dependent transactivation in reporter assays using the direct repeat-4 motif, a binding site of the CAR. This enhancement was synergistically augmented in the presence of steroid receptor coactivator (SRC) 1 or SRC2, both of which are coactivators of the CAR. In addition, TRIM24 was recruited to the CAR-binding element of the CYP2B6 promoter together with the CAR. We also noted that knockdown of TRIM24 suppressed CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells and suppressed CAR-induced CYP3A4 mRNA expression in HepaRG cells but not HepTR/CAR cells. From these results, we suggest that TRIM24 is a novel coactivator of the CAR that is involved in cell- and/or promoter- selective transactivation.

The basal transcriptional activity of unliganded human constitutive androstane receptor (hCAR) was shown to be repressed by the potent liver X receptor (LXR) agonist, TO901317, in a concentration-dependent manner using a reporter assay in cultured cells. TO901317 also repressed the basal transcriptional activity of both mouse and rat CAR. The certified hCAR agonist, CITCO, partially reversed this repressive effect of TO901317 on hCAR basal activity. Unlike hCAR, a three alanine insertion mutant and the splice variant 2 of hCAR require agonists, such as CITCO, to become transcriptionally active and the CITCO-induced reporter activity was repressed by TO901317. As has been previously shown for the typical hCAR inverse agonist, PK11195, TO901317 blocked the interaction of hCAR with steroid receptor co-activator 1 (SRC1). In contrast, the interaction between hCAR and nuclear receptor corepressor 1 (NCoR1) was promoted by PK11195 and TO901317. Furthermore, the hCAR-mediated basal induction of endogenous cytochrome P450 2B6 (CYP2B6) mRNA was adversely affected by co-treatment with TO901317.

Animal defense mechanisms against both endogenous and exogenous toxic compounds function mainly through receptor-type transcription factors, including the constitutive androstane receptor (CAR). Following xenobiotic stimulation, CAR translocates into the nucleus and transactivates its target genes including oxygenic and conjugative enzymes and transporters in hepatocytes. We identified subcellular localization signals in the rat CAR: two nuclear localization signals (NLS1 and 2); two nuclear export signals (NES1 and 2); and a cytoplasmic retention region. The nuclear import of CAR is regulated by the importin-Ran system and microtubule network. Five splice variants (SV1-5) were identified in rat liver in addition to wild-type CAR. When expressed in immortalized cells, their artificial transcripts were inactive as transcription factors. A CAR mutant with three consecutive alanine residues inserted into the ligand-binding domain of CAR showed ligand-dependent activation of target genes in immortalized cells, which is in marked contrast to the constitutive transactivating nature of wild-type CAR. Using this assay system, androstenol and clotrimazole, both of which are inverse agonists of CAR, were classified as an antagonist and weak agonist, respectively. A member of the DEAD box DNA/RNA helicase family (DP97) and protein arginine methyltransferase 5 (PRMT5) were found to be gene (or promotor)-specific coactivators of CAR. The expression of the CAR gene might be under the control of clock genes mediated by the nuclear receptor Rev-erb-α.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Recent studies have reported the anti-tumor effects of the AhR in breast cancer. In this study, we investigated the anti-tumor effect of AhR activation based on the cancer stem cell hypothesis. We show that AhR activation suppressed mammosphere formation of MCF-7 cells and decreased the proportion of cells with high ALDH-1 (aldehyde dehydrogenase 1) activity. In addition, we also demonstrate that AhR activation regulates self-renewal signaling by down-regulating Wnt/β-catenin and Notch.

Human bronchial mucins, such as MUC5AC, have traditionally been defined as a family of high-molecular weight glycoproteins. Changes in the contents of sugar chains on MUC5AC are among the fundamental features in inflammatory respiratory disease. The changes have been shown to lead to unfavorable alterations in the viscosity of mucus, resulting in impairment of mucociliary transport, vulnerability to viral/bacterial infection as sugar chains play an important role in adhesion of some viruses and bacteria to the epithelium, and finally inflammatory cell infiltration in the airway. Recently, we found that expression of some glycosyltransferases associated with the contents and structure of sugar chains is regulated by phosphatidylinositol-phospholipase (PI-PL) C signaling in cells. L-Carbocisteine, a mucoregulatory drug, normalized or balanced fucosylated and sialylated sugar chains, such as sialyl Lewis x through inhibition of PI-PL C signaling. We prepared MUC5AC fusion protein with tandem repeats associated with MUC5AC, and confirmed that L-carbocisteine inhibited the increases in viscosity associated with sialyl Lewis x expression levels. In addition, the clinical study (2008) noted that L-carbocisteine reduced the frequency of common colds and exacerbation of symptoms in patients with COPD. These favorable effects in patients may be due to normalization of sugar chain contents on mucins. We suggest that the inhibitory effect on infection of airway epithelial cells by rhinoviruses, respiratory syncytial virus, and influenza viruses by treatment with L-carbocisteine may also be based on the regulation of sugar chain contents or structures on mucins.

The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. The CAR is normally located in the cytoplasmic compartment of untreated liver cells and translocates to the nucleus after exposure to phenobarbital (PB) or PB-like chemicals. Previously, we identified two nuclear localization signals (NLS) in the rat constitutive androstane/active receptor (CAR), NLS1, which is located in the hinge region, and NLS2, which overlaps with the ligand-binding domain. However, the nuclear import mechanism of CAR is unclear. In this study, we show that nuclear import of CAR is regulated by importin/Ran-GTP systems. The regulation of CAR nuclear import by a Ran-GTP concentration gradient was confirmed using the dominant negative, GTPase-deficient form of Ran (RanQ69L), suggesting the involvement of transport receptors of the importinβ family. IPO13 was shown to be involved in the PB-mediated nuclear translocation of CAR, which was found to be susceptible to inhibition by a dominant negative mutant of IPO13 in primary hepatocytes.

TS-1 is an oral anticancer agent comprising three components: two biochemical modulators of 5-fluorouracil (5-FU) and tegafur (FT), a prodrug of 5-FU. TS-1 displays potent anti-tumor activity by maintaining effective 5-FU concentrations in serum for a prolonged period. FT is gradually converted to 5-FU in vivo via 5’-hydroxylation mediated by cytochrome P450s. As a result, genetic polymorphisms can cause wide individual differences in serum concentration of 5-FU after administration of TS-1, requiring monitoring of serum concentration of 5-FU for each patient. Chemotherapy with TS-1 plays a majo rr ole in the treatment of oral cancer. In cases where a patient with oral cancer shows dysphagia, TS-1 may need to be tube-administered. Although tube administration by the simple suspension method has been recommended fro ma biosafety perspective, particularly for anti-cancer agents, its efficacy remains unclear. We established a simple high-performance liquid chromatography method for simultaneous determination of FT and 5-FU levels at clinical sites by modifying a method reported in the literature. 1) Unlike the original method, this simplified method does not require extraction procedures to separate FT and 5-FU, and requires only 250µ lo f serum. Using this method, we compared F Tc oncentrations in the sera of oral cancer patients administered TS-1 orally or via a tube-assisted simple suspension method. No significant differences in FT concentrations were apparent between these two modes of administration. TS-1 treatment by tube administration using the simple suspension method thus seems useful for patients with dysphagia as an alternative to oral dosage.

The role of the microtubule network in the constitutive androstane receptor (CAR)-mediated transactivation of CYP2B induced by phenobarbital (PB) in rat primary hepatocytes was investigated using the microtubule-disrupting agent nocodazole (NCZ). In human hepatocytes, it was reported that CAR mRNA expression was decreased by a microtubule-disrupting agent through the inhibition of glucocorticoid receptor (GR)-mediated transactivation. However, in the present study, we show that the rat CAR gene was unaffected by the GR-mediated pathway in rat primary hepatocytes treated with NCZ. The PB-induced expression of CYP2B mRNA was repressed in the presence of NCZ for 2 h before and during 4 h of PB treatment, whereas the CAR mRNA and protein expression levels were not affected. Furthermore, the transactivation of the PB-responsive enhancer module-luciferase reporter gene and the nuclear transport of CAR induced by PB were also repressed in the presence of NCZ. Based on these findings, microtubular integrity might be required for PB-induced nuclear translocation of CAR in rat primary hepatocytes. In the same procedures, except that NCZ was replaced with radicicol, the CYP2B mRNA expression induced by PB was also repressed. Taking these into consideration, PB-mediated nuclear translocation of rCAR might be dependent on the 90-kDa heat shock protein as well as the microtubule network.

Vitellogenin (VTG) is a useful biomarker for detecting the estrogenic activity of chemicals in aquatic environments. However, little information is available on the regulatory mechanisms of the expression of each VTG subtype, particularly the relationship between expression patterns of VTG1/2 and estrogen receptor (ER) subtypes, such as ERα and ERβ. In this paper, we measured VTG1 and VTG2 mRNA induction in male medaka liver, which was treated with ERα-selective ligand, (17α, 20E)-3-hydroxy-17,20-[(1-methoxyethylidene)bis(oxy)]-19norpregna-1,3,5(10),20-tetraene-21-carboxylic acid, methyl ester or ERβ-selective ligand, 2-(4-hydroxyphenyl)5-hydroxy-1,3-benzoxazole and investigated the characteristics of ER subtype function in VTG1 and VTG2 inductions. Hepatic VTG1 mRNA was induced by ERα-selective ligands at even low concentration and maximum increases were the same as for E2. VTG2 mRNA was also increased, but its levels were very low. On the other hand, ERβ-selective ligands significantly increased VTG2 mRNA in the presence of ERα agonists. These results indicate that the expression of each VTG subtype is regulated by unique ER subtypes. VTG1 expression is only regulated by the action of ERα. In contrast, VTG2 expression is regulated by both ERα and ERβ, with ERα being essential for VTG2 gene expression and ERβ being essential for enhancement.

Aryl hydrocarbon receptor repressor (AhRR) suppressed, in a ligand independent manner, the ability of estrogen receptor alpha (ERalpha) to enhance the transcription of heterologous estrogen-responsive reporter plasmids in transient transfection assays, as well as of endogenous estrogen-responsive genes in human breast cancer MCF-7 cells. AhRR repressed ERalpha-mediated trans-activation by interfering allosterically with the ligand-independent function of AF-1. The direct interaction between AhRR and ERalpha at the multipartite binding site of ERalpha, which ranges from a DNA binding domain to a ligand binding domain, but did not include the AF-1 moiety was confirmed by a coimmunoprecipitation assay. The AhRR/ERalpha complex was formed in the nuclear compartment and was entrapped by a cis-element in the promoter of E2-responsive genes, as determined in a chromatin immunoprecipitation assay. AhRR might play a role of co-repressor on the transcriptional activity of the ERalpha homodimer.

Control of the intracellular localization of the constitutive androstane receptor (CAR, NR1I3) has more impact on the transcriptional activation of target genes by CAR ligands/activators, including medicines, environmental contaminants and endogenous metabolites, than the conformational change induced by ligand binding, because CAR, unlike other members of nuclear receptor superfamily, is constitutively active. Human CAR (hCAR) and rat CAR (rCAR) were comparatively studied, in primary hepatocytes and in immortal cells, to assess the functional domains controlling nucleo-cytoplasmic shuttling and/or intracellular localization. There are two nuclear localization signals (NLSs) and two nuclear export signals (NESs) in rCAR as well as a cytoplasmic retention region (CRR), whereas hCAR has a single NLS and two NESs. A xenochemical response signal (XRS) controls the neighboring NLS, in a phenobarbital-responsive manner, in both rCAR and hCAR in vivo, but not in immortal cells.

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100–108) and NLS2 in the ligand-binding domain (residues 111–320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220–258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317–358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.

The cyclization-carbonylation of alkynyl hydrazones (I) proceeds readily in the presence of palladous trifluoroacetate in DMSO leading to pyrazole-4-carboxylates (IV) in good to high yields.

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators.

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.

The mammalian constitutive androstane receptor (CAR) is a transcription factor that participates in controlling the expression of xenobiotic metabolizing and transporting genes in response to xenobiotics in an organ-specific manner. In addition to the wild-type CAR (CAR WT) mRNA, mRNAs for five splice variants (SVs) could be detected in the liver of 7-week-old male Wistar rats by RT-PCR using primer pairs covering a full-length mRNA derived from 9 exons; insertion of 18 bp at the 5'-end of intron 8 with or without deletion of 3 bp from the 5'-end of exon 7 (CAR SV1 or SV2), deletion of 4 bp from the 5'-end of exon 8 (CAR SV3), insertion of 195 bp intron 7 (CAR SV4), and insertion of 91 bp intron 6 (CAR SV5). In contrast, only CAR SV5 was detected in lung. Due to the introduction of novel stop codons, all the SVs were considered to code for premature proteins. The liver homogenate gave two protein bands in the vicinity of 37 kDa on Western blotting. They were attributable to CAR WT and SV-complex, respectively, based on their putative molecular weights in descending order. Upon cotransfection with the reporter plasmid, only the cells transfected with the CAR SV4-expression plasmid showed enhanced luciferase activity similar to the WT-transfected cells, for which the further splicing of the remaining intron 7 seemed to be responsible. The transactivation-defective SVs downregulated CAR WT-induced luciferase activity to some extent in the cotransfection experiments.

Both the glucocorticoid receptor (GR) agonist dexamethasone (DEX) and GR antagonist pregnenolone 16α-carbonitrile (PCN) enhanced the transcription of CYP3A1 in rats though in a different fashion. Seven-week old male Wister rats were intraperitoneally administered one to three times with corn oil (vehicle) or DEX and/or PCN (80 mg/kg each) at 24-hr intervals according to various schedules. The animals were sacrificed twenty-four hours after the last treatment, and the dissected livers and lungs were examined for mRNA contents of CYP3A1 and pregnane X receptor (PXR) by real time PCR. DEX appeared to act mainly as an inducer of PXR, that in turn transactivated the CYP3A1 gene, by activating the GR, while PCN was deduced to have a direct effect on the expression of the CYP3A1 gene via activation of PXR from the rapid increase in CYP3A1 mRNA in comparison with DEX. Sequential treatment with DEX and PCN in this order induced CYP3A1 mRNA expression more effectively than treatment in the opposite order. When DEX and PCN were administered simultaneously, the induction of PXR by DEX was reversed by PCN, resulting from their competitive effects on the GR. In the lung, an increase in the CYP3A1 mRNA level was observed in the presence of DEX but not PCN, independently of the GR-activation.

The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to phenobarbital (PB) in rodents. CYP2B forms are highly inducible in liver and intestine in male Wistar rats. In contrast, PB-dependent increases in CYP2B activity are rarely observed in female Wistar rats. The profiles of CAR mRNA expression measured by RT-PCR in various organs in male and female Wistar rats were consistent with those of PB-dependent expression of CYP2B genes. The sex-related dimorphic PB-responses of CYP2B genes in Wistar rats might be determined transcriptionally as are the organospecific PB-responses in both males and females.

In our previous study, chlorinated ethylenes (CEs) such as tetrachloroethylene (PCE), trichloroethylene (TCE) and 1,1-dichloroethylene (1,1-DCE) were found to suppress CYP1A1 activity in rats. In the present study, the effects of CEs on CYP1A1 activity, CYP1A1 mRNA expression and the binding of nuclear proteins to the xenobiotic response element (XRE) were investigated in rat hepatocytes and human hepatoma HepG2 cells. Unexpectedly, CYP1A1 activity was enhanced in the cells cultured in the presence of CEs, suggesting that the suppressive effect of CEs in vivo is indirect. Well consistent with the enhanced enzymatic activity, the expression of CYP1A1 mRNA was increased by all CEs. Furthermore, the induction by 3-methylchoranthrene (3-MC) was reversed in the presence of individual CEs, implying cross-talk between the induction mechanisms of PAHs and CEs. In a luciferase reporter assay of transcription under the control of 4 repeats of XRE, a 2.5-fold increase was induced in HepG2 cells by CEs in the case of 1,1-DCE-treatment, while a 11-fold increase was observed in the cells treated with 3-MC. In HepG2 cells transfected with plasmids expressing aryl hydrocarbon receptor (AhR) antisense mRNA, in which the AhR-expression level was reduced by approximately 40%, no increase in luciferase activity was observed following the CE-treatment, though a 15-fold induction was observed in the presence of 3-MC. As for the nuclear XRE-binding proteins in the presence of CEs analyzed with the gel mobility shift assay, the band-intensity peaked 3 hr after the PCE-, TCE- and 3-MC-treatments. The metabolic activation might be responsible for the 3-hr delay to attain the maximal band-intensity in the case of 1,1-DCE.

The urinary and faecal metabolic profiles of 4bromo-2,5-dimethoxyphenethylamine (2C-B) in rat were investigated. Male Wistar rats were administrated 10 mg/kg of 2C-B hydrochloride orally, and the urine and faeces were collected 0‐24 and 24‐48 hr after administration. The samples were processed by liquidliquid extraction, and the extracts analyzed by GC/ MS, after derivatization. The major metabolite excreted into the urine was 5-O-desmethyl-N-acetyl-2CB, comprising 13.2% of the administrated dose. Other metabolites detected in the urinary extracts were 2-Odesmethyl-N-acetyl-2C-B (5.8%), 2-O-desmethyl-2CB (3.5%), carboxylic acid compound (1.9%), 5-Odesmethyl-2C-B (1.2%) and an alcoholic compound (0.8%). Only 0.2% of the administrated 2C-B was excreted into the urine in its original form. On the other hand, only 5-O-desmethyl-N-acetyl-2C-B and 2-Odesmethyl-N-acetyl-2C-B were detected in the faecal extracts. The major fraction of the urinary metabolites that contained a hydroxy group were recovered as conjugates.

The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to xenobiotics in an organ-specific manner. In the presence of phenobarbital (PB) or PB-type agents, hepatic CYP2B forms are highly inducible. In contrast, PB-dependent increases in the expression of CYP2B activities are rarely observed in the lung. Mature CAR mRNA could be detected in the liver of 7-week-old Wistar rats by RT-PCR, while lung CAR mRNA had a 91 bp extra nucleotide sequence inserted in a coding region of hepatic CAR mRNA. By comparing the full- length sequence of hepatic CAR mRNA, including 5′- and 3′-untranslated region (3′-UTR), with the genomic sequence completed in the present study, the genomic structure was clarified to consist of 9 exons and 8 introns, in which the coding region expanded from exon 2 in close to its 5′-end to the first one-third of exon 9 after 159 bp of 5′-UTR in the most frequently obtained cDNA clones. In pulmonary CAR mRNA, intron 6 was not spliced out, implying that the lack of CAR in the lung might in part result from the incomplete splicing of precursor mRNA during its maturation.

The arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells was employed in a longitudinal study of pollution in the Sagami River flowing into Sagami Bay using river sediment extracts. The six sampling points for river sediment were Ogurabashi, Shimomizo, Atsugi, Kurami, Shinomiya, and Hiratsuka (in descending order of distance from the estuary). In samples obtained at the four midpoints along the river, ECOD activity was induced almost to the same extent. Ogurabashi, the point farthest from the estuary, seemed less polluted than downstream areas based on ECOD activity, while a marginal increase in the activity above the baseline was found at the highest concentration in the sample from Hiratsuka. The points where the river sediment caused a marked induction of ECOD activity in HepG2 cells were surrounded by inland industrial areas scattered along the river.

The pollution level of the Tsurumi River flowing into Tokyo Bay was studied longitudinally using river sediment extracts in an assay system based on the arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The sampling points of river sediment were as follows: Namamugi, Kami-Sueyoshi, Tsunashima, Shin-Yokohama A and B (downstream and upstream, respectively, from the former open-air industrial waste incineration site), Kozukue and Nakayama (Fig. 1). ECOD activity was induced to different extents in all samples tested. Namamugi, located in the middle of Keihin Industrial District, was considered to be the most polluted, followed by Shin-Yokohama A and Tsunashima. Results from these samples showed reverse U-shaped dose-response curves in terms of ECOD activity as well as 3-methylcholantrene (3-MC) although the expression of CYP1A1 mRNA in HepG2 cells remained roughly constant at higher concentrations as in the case of 3-MC, thus implying post-transcriptional suppression.

Chlorinated ethylenes (CEs) are known to be metabolized by CYP2C as well as CYP2E1 and CYP2B. The effects of CEs on the expression of the rat CYP2C gene in the liver and lung were comparatively studied in terms of the enzyme activity, and protein and mRNA contents. Tetrachloroethylene (PCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE) were injected at 0.5 g/kg i.p. into male 7-week-old Wistar rats individually or in combination with phenobarbital (PB). The expression of CYP2C mRNA showed organ-specific properties without being detectable in the lung under the assay conditions employed in the present study. Individual CEs had no effect on the enzymatic activity of CYP2C estimated by 2α-hydroxylation of testosterone (2α-TSH), whereas PB markedly enhanced the enzymatic activity in the liver but not the lung microsomes, although it was highly susceptible to the suppressive effect of 1,1-DCE. A down-regulation of pulmonary enzyme activity was observed with PCE by an unknown mechanism when the animals were treated simultaneously with PB. 1,1-DCE was found to inhibit the expression of hepatic mRNA irrespective of the coexistence of PB, and the same was true for CYP2C protein in the same organ to a lesser extent. In an analogy to our previous findings with other xenobiotic-metabolizing CYP forms such as CYP2B1/2 and CYP2E1, a potent suppressive effect of 1,1-DCE was observed with the maximum around 18 hr after the treatment on both constitutive and PB-induced expression of CYP2C.

Chlorinated ethylenes (CEs) including tetrachloroethylene (PCE), trichloroethylene (TCE) and 1,1-dichloroethylene (DCE) were comparatively evaluated for their effects on the expression of cytochrome P450 (CYP) forms of subfamilies 1A, 2B, 2E and 3A as well as their relative suitability as substrates of these CYPs. The magnitudes of inhibition of the enzyme activities were as follows in descending order: 1,1-DCE > TCE > PCE against hepatic CYPs and PCE > 1,1-DCE > TCE against pulmonary CYP2B1. These organ-specific profiles in the sensitivities to the adverse effects of CEs were partly attributable to the differential expression patterns of CYP forms by which they were metabolically activated. The expression of hepatic and pulmonary CYP2B mRNA was severely suppressed in the presence of 1,1-DCE during the entire observation period until 30 hr after the CE-treatment, in marked contrast to the temporarily enhanced expression at 6 hr followed by a moderate suppression in the cases of PCE and TCE with the trough values being observed at 18 hr. In addition to CYP2B, 1,1-DCE in advance of the transcriptional stage, when simultaneously treated with phenobarbital, also exclusively suppressed CYP2E1. These general suppressive effects of 1,1-DCE on the expression of divergent CYP mRNAs in vivo resembled the published findings in primary cultured hepatocytes treated with inflammatory cytokines such as IL-1β, TNF-α and IL-6, implying the highly inflammatory nature of 1,1-DCE.

Cyclization–carbonylation of α,β-alkynic hydrazones and (o-alkynylphenyl) (methoxymethyl) sulfides with Pd(tfa)2 in DMSO/MeOH afforded methyl pyrazole-4-carboxylates and benzo[b]thiophene-3-carboxylates, respectively, in good yields. A simple change of the ligand (solvent) allowed controlled, effective switching between cyclization–carbonylation–cyclization-coupling (CCC-coupling) reactions and cyclization–carbonylation reactions.

Three polychlorinated ethylenes (CEs), tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (DCE), were comparatively studied for their effects on the expression of cytochrome P450 (CYP) mRNA forms in the liver and lung using 7-weeks-old male Wistar rats. The animals were i.p. inoculated with 0.5 g/kg of the individual CEs and sacrificed at 6-hr intervals for 30 hr for the determination of the mRNA levels of hepatic and pulmonary CYP2B and hepatic CYP2E1. A 3.5-fold increase in the expression of hepatic CYP2B mRNA was noted transiently at 6 hr in the presence of PCE. In contrast, 1, 1-DCE was suppressive and started within 6 hr and lasted for more than 30 hr, with a trough value of 15% of the control being observed around 12 to 18 hr, while there was no marked effect observed in the case of TCE-treatment. The expression of pulmonary CYP2B mRNA was severely suppressed in the presence of 1, 1-DCE during the entire observation period as was the case with its hepatic counterpart, while the temporarily enhanced expression of mRNA at 6 hr by PCE and TCE was followed by a moderate suppression, with the trough values of ca. 80 and 65% of the control, respectively, at 18 hr. Concerning the hepatic CYP2E1, the expression of mRNA was adversely affected by all the CEs with a descending order of magnitude of 1, 1-DCE, TCE and PCE with peak inhibition values of 85%, 50% and 35%, respectively. The nonselective suppressive effect of 1, 1-DCE on the expression of divergent CYP mRNAs was well correlated with the IL-1β-dependent suppression of various types of CYP mRNA in primary cultured hepatocytes previously reported. Although PCE and TCE are under stringent legislative restriction in Japan as environmental pollutants owing to their relatively stable natures, they are less toxic in terms of the inhibition of the mRNA expression of CYP forms in acute phases than short-lived 1, 1-DCE, which is inflammatory to the host animals; the shorter the environmental life span (1, 1-DCE > TCE > PCE), the more severe the acute toxicity.

The in vitro inhibitory effects of heavy metals, i.e., mercury, cadmium, lead, nickel and beryllium in their chloride forms, on rabbit pulmonary drug-metabolizing enzymes were studied comparatively. Microsomal and cytosolic fractions prepared from rabbit lungs were incubated in the presence of heavy metals prior to enzyme assays. Mercury was the most potent in reducing cytochrome P450 content and mixed-function oxidase activities including NADPH-cytochrome c reductase and benzo[a]pyrene hydroxylase. The addition of mercury to pulmonary microsomal preparations resulted in a spectral shift from 450 nm to 420 nm in an absorption maximum of cytochrome P450carbon monoxide complex with mercury chloride being more potent than methylmercury. Cadmium was also inhibitory, while the effects of nickel were noted only at higher concentrations. Neither lead nor beryllium was inhibitory. Among second-phase drug-metabolizing enzymes, UDP-glucuronyltransferase and glutathione S-transferase were found to be susceptible to the adverse effects of mercury and lead, respectively. The extent of inhibition of the latter activity by mercury were highly dependent on the concentration of glutathione, implying the complex formation between them. Cadmium was slightly inhibitory to both enzyme activities, though the other metals had no effect. The results indicate that the pulmonary toxicities of airborne heavy metals could be inferred using simple in vitro assays.

The extent of pollution in the rivers flowing into Tokyo Bay was studied comparatively using river sediment extracts in an assay system based on the arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The sampling points of river sediment were as follows: Namamugi on the Tsurumi River, Rokugo on the Tama River, Hirai, Senjyu and Funado on the Ara River, Baraki on the Edo River, and Makuhari on the Hanami River (Fig. 1). The Tsurumi River (Namamugi) located in the middle of Keihin Industrial District was considered most polluted, followed by the Ara River (Hirai). The river sediment collected at Baraki on the Edo River was least polluted. The extracts of highly polluted river sediments sampled at Namamugi and Hirai exhibited a reverse U-shaped dose-response curve, for which polycyclic aromatic hydrocarbons (PAHs) might be mainly responsible, in a range of non-toxic concentrations. The pollution of river sediment collected at Namamugi was roughly estimated to be 2 to 20 μg as PAHs/g sediment by consulting the data of Nakama et al. and 0.2 to 2 μg as PAHs/g was assigned to the river sediment of Hirai.

The manifestation of toxicity by certain environmental compounds requires enzymatic activation. It is well known that chlorinated ethylenes (CEs) are metabolized by intracellular enzymes represented by cytochromes P450 (CYPs) to the urinary secreted forms via epoxide intermediates, which are responsible for their toxicities by forming complexes with intracellular components including CYPs. In order to study the roles of CYPs in the metabolic activation of tetrachloroethylene (PCE), trichloroethylene (TCE) or 1, 1-dichloroethylene (1, 1-DCE), the cytotoxicity of individual CEs were tested in primary hepatocyte cultures established from animals treated with various CYP-inducers such as 3-methylchoranthrene (inducer of CYPlAl/2), phenobarbital (CYP2B1/2) and pyridine (CYP2E1). The cytotoxicity of CEs measured by lactate dehydrogenase leakage after 24 hr was enhanced in different fashions, depending on the CYP inducers used. The results are summarized as follows: CYP1A1/2 and 2B1/2 raised the cytotoxicity of PCE;CYP2B1/2 was exclusively associated with the enhanced cytotoxicity of TCE; and CYP2E1 and 2B1/2 were proved to potentiate 1, 1-DCE. Based on these observations, CEs differing in the number of chlorine substitutions were disclosed to have divergent preferences for CYPs, by which they were metabolically activated.

Many environmental chemicals (xenobiotics), including polychlorinated ethylenes (CEs), are known to be metabolically intoxicated via the formation of hazardous intermediates (referred to as biological activation), while the expression of metabolic enzymes might be interfered with by xenobiotics at various stages in a system to minimize their adverse biological effects on the host animals. Three CEs, i.e., tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (1, 1-DCE), were comparatively studied for their effects on the in vivo expression of CYP forms, which are responsible for their metabolic activation, as well as organospecificities between the liver and lung. Furthermore, their effects on the expression of CYP forms were studied in the animals simultaneously administered with phenobarbital (PB), known as an inducer of CYP2B. Individual CEs were administered intraperitoneally at 0.5 g/kg alone or simultaneously with PB (80 mg/kg/d) to 7-week-old male Wistar rats weighing about 200 g. The testosterone hydroxylase activities, CYP2B- and 2E1-mRNA, and CYP2B- and 2E1-proteins were measured 24 hr after the treatment. Testosterone 2β-hydroxylase (2βTSH) and 16βTSH activities are attributable to the functions of CYP3A2, and CYP2B plus CYP3A2, respectively. The induction of hepatic CYP2B in the PB-treated animals might be masked by the suppressive effect of CYP3A2 in terms of the 16βTSH activity, while the pulmonary 16βTSH activity was confined to the function of CYP2B, which was insensitive to the inducing effect of PB. The inhibition of hepatic 16βTSH activity was observed exclusively in the presence of 1, 1-DCE, especially in PB-coadministered animals, suggesting the preferable suppression of CYP2B. In the lung, however, PCE suppressed the 16βTSH activity in the absence of PB. The expression levels of hepatic CYP2B mRNA and protein were significantly lowered by 1, 1-DCE in the presence of PB. Concerning hepatic CYP2E1, no alternation was observed in the levels of mRNA and protein by any of the CEs in the absence or presence of PB, except for a marked decrease in the amount of mRNA when the rats were treated with 1, 1-DCE in combination with PB. In the lung, both mRNA and protein were not detected under the given assay conditions, as in our previous studies. Based on these results, it is suggested that 1, 1-DCE suppresses the induction of hepatic CYP2B and 2E1 in advance of the transcriptional stage. The expression of pulmonary CYP2B was obstructed by PCE posttranslationally in the absence of PB.

Almost all of fucose and sialic acid in mucus are found on the mucus glycoproteins (mucins), and these sugar components on mucins are known to be associated with the viscous property of mucus. We have reported some aspects of carbocisteine, a mucoregulatory drug, correcting fucose and sialic acid contents in mucus. At present, carbocisteine's expectorant action of airway mucus is postulated to involve - the regulation of fucose and sialic acid contents on mucins. However little information is available about the relationship between the viscosity and sugar contents on mucins when treated with carbocisteine. To investigate further the mechanism behind the action of carbocisteine, the present study prepared MUC5AC fusion protein which has tandem repeat regions associated with MUC5AC, and evaluated the effects of carbocisteine on tumor necrosis factor (TNF)-alpha-induced increases of mucus viscosity and sialyl-Lewis x-epitopes antigen, an antigen which consists of fucosylated and sialylated sugar chains on the MUC5AC fusion proteins. Carbocisteine inhibited the TNF-alpha-induced increases of the viscosity and sialyl-Lewis x-epitopes on MUC5AC fusion protein. These findings suggest that carbocisteine may normalize the viscosity of mucus through "balancing" of fucose and sialic acid contents on airway mucins.

The efficacy of the extraction of polychlorinated biphenyls (PCBs) from varnish-infiltrated insulating papers as a model of solid materials with an aqueous sodium hydroxide (NaOH) by autoclaving at 121°C for 30 min was compared with those for the conventional procedures, i.e., saponification with 1 N ethanolic NaOH in a boiling water bath for 60 min and extraction with benzene in a Soxhlet apparatus. The newly invented autoclaving method was found to be superior to the conventional ones, yielding approximately 5- to 6-fold cumulative PCB congeners without being accompanied by extended decomposition. Therefore, the autoclave-based sample treatment is recommended for more accurate determination of PCBs in the case of PCB-impregnated solid materials such as hardened oils and resin-coated or -infiltrated papers instead of being treated conventionally.

Natural water quality became worse these days by producing more pollutants than the amounts of decomposition in nature by primary decomposers. So, photocatalyst was intended to improve the environmenal water quality. However, photocatalyst was known to act both the oxidative decomposition of pollutants as well as primary decomposers. In order to know whether photocatalyst can contribute to the net result for the improvement of environmental water quality or not, phtocatalyst was applied to model polluted water. The rate of purification was measured by the rate of consumption of dissolved oxygen. When photocatalyst was applied, dissolved oxygen was consumed 5.7 times faster under bright solar irradiation, 3.3 times faster at twilight time, and 1.5 times faster even at night, than in model environmental polluted water without photocatalyst. These results show that photocatalyst can improve polluted water efficiently, if water can supply enough air, even in the presence of primary decomposers in both day and night.

Chlorinated ethylenes (CEs) such as tetrachloroethylene (PCE), trichloroethylene (TCE), 1, 1-dichloroethylene (1, 1-DCE), cis-1, 2-dichloroethylene (cis-DCE), and trans-1, 2-dichloroethylene (trans-DCE) are members of the class of volatile halogenated hydrocarbons. Each was administered intraperitoneally at 0.5g/kg alone or simultaneously with phenobarbital (PB, 80mg/kg/d) to male Wistar rats weighing about 200g (7-weeks-old). Microsomal fractions of livers and lungs removed from animals sacrificed 24h after treatment were tested for monooxygenase activities and protein content of 4 cytochrome P450 (CYP) forms, i.e., CYP1A1/2, 2B1/2, 2E1 and 3A1/2. In terms of constitutive expression, they were compensatory in liver and lung with CYP1A1/2, 2E1 and 3A1/2 being detected only in liver and CYP2B1 only in lung. All 5 CEs employed in this work suppressed hepatic CYP1A1/2, 2E1 and 3A1/2 in the descending order of 1, 1-DCE > cis-DCE > trans-DCE > TCE > PCE. The magnitude of suppression of pulmonary CYP2B1 by CEs were compared as follows; PCE > trans-DCE > 1, 1-DCE > cis-DCE > TCE. CYP1A1/2, CYP2E1 and CYP3A1/2 in hepatic microsomes from PB-treated animals responded to CEs similarly to those from PB-untreated animals. These 3 enzyme activities could not be detected in pulmonary microsomal fractions, irrespective of the PB-treatment. Hepatic CYP2B1/2 could be detected only when treated with PB in marked contrast to the constitutive expression of pulmonary CYP2B1. The suppression of PB-induced hepatic CYP2B1/2 was observed with all 5 CEs, with special emphasis on complete inhibition with 1, 1-DCE. The adverse effects of TCE and PCE on pulmonary CYP2B1 from PB-treated rats were comparable with those from PB-untreated animals. In contrast, 3 dichloro-isomers were more suppressive to the enzyme in the absence than in the presence of PB. The protein levels of hepatic CYP forms were generally proportional to the enzyme activities in the case of 1, 1-DCE-treatment. The amounts of pulmonary CYP2B1 apoprotein were in good accordance with the enzyme activities when treated with CEs individually.

Trichloroethylene (TCE) is a highly toxic compound which belongs to the class of volatile halogenated hydrocarbons, which are well-known pollutants of drinking water and the atmosphere. The effects of moderately toxic concentration of TCE on the levels of cytochrome P450 (CYP) isoforms and on their phenobarbital (PB) induction in rats were studied. Monooxygenase activities associated with individual CYP isoforms, i.e., CYP1A, 2B, 2E1 and 3A, were measured in microsomal fractions prepared from both lungs and livers of male and female Wistar rats (7-weeks old). Differences in constitutive levels of CYP isoforms were observed between liver and lung of mock-treated rat ; microsomal CYP2B activity was solely detected in lung while the activities of CYP1A, 2EI and 3A, but not 2B were detected in liver. Among these, the pulmonary CYP2B and hepatic CYP2E1 activities were reduced by TCE-treatment. PB-treatment resulted in the detection of increased levels of hepatic CYP1A and 2E1 and of pulmonary CYP2B activities, and also the appearance of hepatic CYP2B activity. Coadministration of TCE was suppressive against the activities of CYP isoforms except for hepatic CYP2E1 in PB-treated rats. The lowered CYP2B activity in the presence of TCE was accompanied by the reduction in the amount of CYP2B apoprotein.

Organic halogens (OX) extractable with a water-miscible organic solvent in samples from municipal solid waste incinerators (MSWIs) were determined quantitatively using apparatus for measuring total organic halogens in drinking water. Besides OX, polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) in samples from MSWI were analyzed separately to compare their behavior in MSWI. OX was generated both in flue gas and the electrostatic precipitator and exhibited a linear correlation with both PCDD and PCDF, implying that PCDD and PCDF were derived from OX. These results show that OX is a useful surrogate marker for PCDD and PCDF in MSWIs.

The effect of wearing of sand in the furnaces of fluidized bed incinerators (FBIs) on the formation of polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF) and their related compounds from polychlorinated biphenyl (PCB) and 1,2,3-trichlorobenzene (1,2,3-T3CBz) was studied experimentally using a pyrolysis apparatus consisting of an injection unit; a stainless steel tube, in which packed was either unused sand (virgin sand) or used for two months in FBI (worn-out sand), heated to 650°C by an electric furnace; and a sampling unit. Furthermore, polychlorinated diphenyl ether (PCDE) was pyrolyzed on worn-out sand in order to compare the formation mechanisms of PCDD and PCDF with those from PCBs. Besides the generation of PCDD and PCDF, the hydroxylation of the starting compounds occurred. PCB was detected in the pyrolyzed samples of 1,2,3-T3CBz on both types of sand. In all cases, PCDF was in much higher yield than PCDD and pyrolysis on worn-out sand was more favorable for the formation of PCDD, PCDF and their relatives than pyrolysis on virgin sand. There was marked difference in metal concentrations between virgin and worn-out sand, implicating that the increased metal concentrations in sand would promote the formation of PCDD, PCDF and their related compounds in FBIs.

In a fluidized bed incinerator (FBI), municipal solid waste incinerated with the help of sand which had been repeatedly used for two months (worn-out sand) yielded one-order higher concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated benzenes (PCBzs) and polychlorinated phenols (PCPs) in comparison with that incinerated on unused (fresh) sand. As metal concentrations in sand increased after each cycle of incineration, the worn-out sand used contained one to two-order higher amount of metals than the sand used for the first time. Taken together, these findings might imply that the accumulation of metals in worn-out sand is responsible for the increase in the amounts of polychlorinated compounds.

The risk of daily exposure to volatile halogenated hydrocarbons (VHH) in ambient air was assessed by the urinary concentration of VHH metabolites. Thirty residents in the urban Tokyo area, 13 males and 17 females, aged at 7 to 78, were enrolled in the present study. They were requested to carry passive gas samplers adsorbing chemicals in the ambient air during daily activities. The mean levels (mean±S.D.) of 1, 1, 1-trichloroethane (1, 1, 1-TCE), trichloroethylene (TCE) and tetrachloroethylene (PCE) in the breathing air for 12 h were 3.4±2.4μg/m3, 2.0±1.3μg/m3 and 1.1±0.8μg/m3, respectively. The daily intake of individual VHH was calculated to be 68, 40 and 23μg/person for 1, 1, 1-TCE, TCE and PCE, respectively, while women inhaled twice as much VHH as men. Since total VHH levels in the breathing air correlated well with the levels of urinary metabolites (γ=0.80), the latter could be employed as a biological index representing the level of exposure to VHH.