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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental contaminants such as dioxins and polycyclic aromatic hydrocarbons. Following ligand binding, AhR binds to xenobiotic responsive elements and modulates the transcription of AhR target genes. Multiple studies have shown that AhR plays important roles in a range of cancer cells and is attracting attention as a therapeutic target for cancer treatment. We have previously reported that AhR agonists inhibit tumorsphere formation in an AhR-dependent manner in the MCF-7 breast cancer cell line. In the present study, we found that FDI-6, an inhibitor of the transcription factor Forkhead Box M1 (FOXM1) induced the mRNA expression of AhR target genes, nuclear translocation of AhR, and transcriptional activity of AhR. In addition, FDI-6 dose-dependently reduced the mRNA expression of FOXM1-regulated genes in AhR-expressing MCF-7 cells, although not in AhR-deficient MCF-7 cells. Furthermore, FDI-6 was found to suppress tumorsphere formation via the AhR in MCF-7 cells and HepG2 human liver cancer cell line. On the basis of the findings of this study, we show that FDI-6, a FOXM1 inhibitor, functions as an AhR agonist, and suppresses tumorsphere formation via the AhR.

Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress–activated Chk1 or Chk2, which triggers its SCFβ-TrCP–mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress–induced cell cycle checkpoint. [Cancer Res 2009;69(16):6438–44]

Supplementary Figure 1 from Stress-Activated Mitogen-Activated Protein Kinases c-Jun NH2-Terminal Kinase and p38 Target Cdc25B for Degradation

Supplementary Materials from Stress-Activated Mitogen-Activated Protein Kinases c-Jun NH2-Terminal Kinase and p38 Target Cdc25B for Degradation

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.

Breast cancer is the most commonly diagnosed cancer among women worldwide. Despite a variety of drugs available for the treatment of patients with breast cancer, drug resistance remains a significant clinical problem. Therefore, there is an urgent need to develop drugs with new mechanisms of action. Camalexin is the main indole phytoalexin in Arabidopsis thaliana and other crucifers. Camalexin inhibits the proliferation of various cancer cells. However, the mechanism by which camalexin inhibits cell proliferation remains unclear. In this study, we found that camalexin inhibited cell proliferation and migration of breast cancer cell lines. Furthermore, camalexin also suppressed breast cancer stem cell-derived mammosphere formation. We previously reported that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) agonist suppresses mammosphere formation. Several compounds with indole structures are known to act as AhR agonists. Therefore, we hypothesized that the inhibition of mammosphere formation by camalexin may involve AhR activation. We found that camalexin increased the nuclear translocation of AhR, AhR-mediated transcriptional activation, and expression of AhR target genes. In addition, camalexin suppressed mammosphere formation in AhR-expressing breast cancer cells more than in the breast cancer cells that lacked AhR expression. Taken together, the data demonstrate that camalexin is a novel AhR agonist and that the inhibition of cell proliferation, migration, and mammosphere formation by camalexin involves the activation of AhR. Our findings suggest that camalexin, an AhR agonist, may be a novel therapeutic agent for breast cancer.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

Triple-negative breast cancer (TNBC) is associated with poor prognosis, because of no effective targeted therapy. In the present study, we demonstrated the crucial role of the aryl hydrocarbon receptor (AhR) in mediating the effects of the chemotherapeutic agent doxorubicin (DOX) in the chemotherapeutic sensitivity of TNBC. Firstly, we established AhR knockout (KO) MDA-MB 231 TNBC cells. The cytotoxic effects of DOX were more pronounced in AhR KO cells than in parental cells. In addition, our results indicated that AhR KO cells showed downregulated expression of DOX-metabolism enzyme, aldo-keto reductase (AKR) 1C3, relative to those of parental cells. Furthermore, AhR was found to enhance AKR1C3 promoter reporter activity, suggesting that AKR1C3 mRNA transcription is activated by AhR. Additionally, our findings confirmed that the downregulation of AKR1C3 expression enhanced DOX sensitivity in MDA-MB 231 cells. Finally, AhR and AKR1C3 expression were positively correlated in human breast cancer. Taken together, our results suggested that AhR is involved in DOX sensitivity by regulating AKR1C3 expression in TNBC cells.

The aryl hydrocarbon receptor (AhR) regulates expression of genes encoding drug/xenobiotic metabolizing enzymes. Cytochrome P450 (CYP) 3A5 is involved in drug metabolism. However, regulation of CYP3A5 gene expression is not yet well understood. In this study, we aimed to investigate the effect of the ligands of AhR on CYP3A5 gene expression. CYP3A5 mRNA expression was induced by the polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3MC) and benzo[a]pyrene in HepG2 cells. To determine whether the PAHs induced CYP3A5 gene expression via AhR, we generated AhR knockout (AhR KO) HepG2 cells. CYP3A5 mRNA expression was not induced by 3MC treatment in AhR KO cells. In addition, we generated AhR rescue cells from AhR KO cells and evaluated CYP3A5 mRNA expression. We found that CYP3A5 mRNA expression was induced by 3MC treatment in AhR rescue cells. Taken together, these results demonstrated that CYP3A5 mRNA expression was induced by PAHs via AhR in HepG2 cells. Our findings suggest that ligand-activated AhR affects CYP3A5-mediated drug metabolism.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets.

Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription.

金沢大学医薬保健研究域薬学系, Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFβ-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint. ©2009 American Association for Cancer Research.

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin– induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5α-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element–driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT. Keywords:: androgen receptor, aryl hydrocarbon receptor, cross-talk, complex formation, transcriptional activation

Estrogenic/antiestrogenic activities of 14 polycyclic aromatic hydrocarbons (PAHs) and 63 monohydroxylated PAHs (OHPAHs) having 2 to 6 rings were evaluated by yeast two-hybrid assay expressing human estrogen receptor α. Relative effective potencies of estrogenic and antiestrogenic activities were calculated as the inverse values of the relative concentration of the test compound that gave the same activities of E2 and 4-hydroxytamoxifen, respectively. PAHs did not show any estrogenic/antiestrogenic activity, but several OHPAHs having 3 to 5 rings showed activities. Especially, OHPAHs having 4 rings such as 3-, 4- and 10-hydroxybenz[a]anthracenes (3-, 4- and 10-OHBaAs) and 2-hydroxychrysene (2-OHCh) showed strongly estrogenic activity. Several other OHPAHs having 4 rings such as 2- and 3-hydroxybenzo[c]phenanthrenes (2-, 3- OHBcPhs), 2-OHBaA and 3-OHCh showed strongly antiestrogenic activity. The length-to-breadth (L/B) ratios of the rectangular van der Walls planes surrounding the ring molecules of estrogenic OHPAHs were in the narrow range from 1.599 to 1.734. The distances between the oxygen atom of the phenol group and farthest hydrogen atom (O-H distance) of the estrogenic OHPAHs ranged from 10.825 A° to 11.738 A°. The L/B ratios and O-H distances of antiestrogenic OHPAHs were in the wider ranges from 1.277 to 1.734 and from 8.47 A° to 11.681 A°, respectively. The partial charges (atomic unit) of the phenol group of both estrogenic and antiestrogenic OHPAHs were in the range from 0.250 atmic unit (au) to 0.253 au. The similarity of these values to those of E2 and diethylstilbestrol suggested that the compositions of estrogenic OHPAHs were similar to them and that the compositional conditions of estrogenic OHPAHs were much smaller than those of antiestrogenic OHPAHs. These results raise the possibility of predicting the estrogenic/antiestrogenic activities of OHPAHs from their structural characteristics, although using only the above three parameters might not be enough for accurate estimations.

金沢大学ナノ生命科学研究所 / 金沢大学大学院医学系研究科機能分子医薬学, Estrogen receptor α (ERα) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERα gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERα proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V), and G1231deletion (411fsX7). The mutated ERα expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ERα proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERα proteins except ERα (G160C) and ERα (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERα (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERα (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERα expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10 pM-100 nM E2) of the mutant ERα proteins except ERα (E353V) and ERα (411fsX7) were similar to that of wild-type ERα. However, at a low level of E2 (100 pM), the mutants ERα (N69K), ERα (L296P), ERα (S309F), and ERα (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERα. The mutants ERα (E353V) and ERα (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERα (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERα mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens. © 2006 Elsevier Ltd. All rights reserved.

Epidemiological studies suggest that diesel exhaust particles (DEP) contribute to an increase in allergic diseases. To assess the effects of DEP on the central immune system, mice were exposed to DEP by intraperitoneal (IP) administration. Exposure to DEP resulted in severe thymic involution accompanied by a reduction in the number of thymocytes, especially in cortical CD4+CD8+ double positive and double negative subsets. Core carbon particles associated with a mixture of chemical compounds in DEP did not appear to be responsible for the DEP-induced thymic involution because carbon graphite does not affect neither the number nor the CD4/CD8 profile of thymocytes. Extraction of DEP by ether, acidic and basic solvents showed that several independent fractions including the neutral ether fraction, which contains polycyclic aromatic hydrocarbons (PAH), induced thymic involution. Among major PAH components of DEP, benzo[b]fluoranthene (BbF), benzo[a]pyrene (BaP), and benzo[k]fluoranthene (BkF) were very potent...

Airborne particulates were collected in seven cities in the Pan–Japan Sea countries, Shenyang (China), Vladivostok (Russia), Seoul (South Korea), Kitakyushu, Kanazawa, Tokyo and Sapporo (Japan), in winter and summer from 1997 to 2002. In addition, particulates from domestic coal-burning heaters and diesel engine automobiles were collected in Shenyang and Kanazawa, respectively. Nine polycyclic aromatic hydrocarbons (PAHs) and four nitropolycyclic aromatic hydrocarbons (NPAHs) in the extracts from the particulates were analysed by HPLC with fluorescence and chemiluminescence detections, respectively. The PAHs were fluoranthene, pyrene (Pyr), benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene, and NPAHs were 1,3-, 1,6-, 1,8-dinitropyrenes, and 1-nitropyrene (1-NP). Mean atmospheric concentrations of PAHs in Shenyang and Vladivostok were substantially higher than those in Seoul, Tokyo, Sapporo, Kitakyushu and Kanazawa. However, the mean atmospheric concentrations of NPAHs were at the same levels in all cities except Kitakyushu. The expected seasonal variations (greater PAH and NPAH concentrations in winter than in summer) were observed in all cities. In order to study the major contributors of atmospheric PAHs and NPAHs, both cluster analysis and factor analysis were used and three large clusters were identified. Furthermore, the concentration ratios of 1-NP to Pyr were significantly smaller in Shenyang, Vladivostok and Kitakyushu and the values were close to those observed in particulates from coal stove exhaust. By contrast, in Seoul, Kanazawa, Tokyo and Sapporo the [1-NP]/[Pyr] ratio reached values similar to those of particulates released from diesel-engine automobiles. The [1-NP]/[Pyr] concentration ratio seemed to be a suitable indicator of the contribution made by diesel-engine vehicles and coal combustion to urban air particulates.

A new dinuclear docking Pt(II) complex, (cis-diammine) (l-1,2-cyclohexanediamine)(mu-dichloro)-diplatinum(II) oxalate was synthesized by reacting oxaliplatin(l-OHP, [Pt(oxalato)(L-dach)]), L-dach = 1R, 2R-cyclohexanediamine), with cisplatin (CDDP). Elemental analysis of the compound indicated that it was 1:1 molar ratio complex of oxaliplatin and cisplatin. A plausible chemical structure has been proposed as Cl(-) bridged dinuclear complex, judged from its yellow coloration and NMR spectral analysis. This complex can be denoted as, i.e. [Pt(2)Cl(2)(NH(3))(2)(L-dach)](COO)(2) (L-OHP/CDDP). The complex showed higher cytotoxicity against L1210 than the parent complexes and low cross-resistance against L1210/CDDP and L1210/DACH. Its antitumor activity was also tested in vivo against murine leukemia L1210 cell lines. The complex showed much higher activity than the mixture(1:1 molar ratio) of oxaliplatin and cisplatin. The antitumor effect against L1210/CDDP was very high, showing collateral sensitivity, being similar to that of oxaliplatin, and against L1210/DACH it showed no cross-resistance.

We investigated the influences (cell toxicity, endocrine disrupting action, etc.) of polyaromatic hydrocarbons (PAHs) and heavy metals on the human thyroid gland by using a cultured thyroid cancer cell (8505C). Among the six PAHs tested (anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[k]fluoranthene, chrysene and pyrene), anthracene did not show any effect, but cell proliferation was enhanced by benzo[a]anthracene, benzo[a]pyrene and benzo[k]fluoranthene. Benzo[a]pyrene had an especially marked effect. On the other hand, cell toxicity was exhibited in four (cadmium, copper, nickel and zinc) of the twelve metals tested (aluminum, cadmium, calcium, copper, lead, magnesium, manganese, nickel, tin, zinc, selenate and selenite), and in particular, the influence of cadmium was remarkable. It was found that the toxicity of cadmium was decreased by the addition of high concentrations of either calcium or selenite.

金沢大学大学院自然科学研究科環境創成, 金沢大学薬学部, In recent years, there have been many attempts to reuse molten slag. Molten slag is obtained through further treatment of incinerated ash produced in the disposal process of garbage or sewage sludge and is used in building materials, such as brick. To establish the safety of such slag, its physical properties and metal contents were investigated in this study. After examining the physical and chemical characteristics of 13 forms of slag made using different methods, no differences were observed and it was judged that reuse is possible. There was little elution of toxic metals in the elution test when water was used as a solvent. On the other hand, when acid and alkali were used, the elution of toxic metals tended to become greater. © 2004 The Pharmaceutical Society of Japan.

To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each. The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells. While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed. In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and β-naphthoflavone. Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load. In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities.

We examined endocrine disruptors in diesel exhaust particulates (DEP) emitted from a diesel engine under different engine loads. The antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activity of DEP extracts (DEPE) were evaluated using PC-3/AR human prostate cancer cells transiently transfected with androgen- or AhR agonist-regulated luciferase reporter plasmids, respectively. The antiandrogenic and AhR agonistic activities were enhanced by higher engine load. The AhR antagonist α-naphthoflavone reversed the antiandrogenic activity of DEPE as seen through a reduction in AhR agonistic activity. The concentrations of polycyclic aromatic hydrocarbons (PAHs) with four or more rings, which are AhR agonists in DEPE, increased with higher engine load. Therefore, the enhanced antiandrogenic activity of DEPE is due in part to the increase in AhR agonists such as PAHs with higher engine load.

2-Hydroxyfluorene (2-OHF) and 1-hydroxypyrene (1-OHP) in urine samples of smokers and nonsmokers were determined separately by using two high-performance liquid chromatography (HPLC) systems with fluorescence detection. Urine samples were collected from the subjects who lived in Japan and Thailand, and were not occupationally exposed to polycyclic aromatic hydrocarbons (PAHs). The mean concentrations of 2-OHF and 1-OHP of Thai smokers (0.75 and 3.03 μmol/mol creatinine) and nonsmokers (0.22 and 0.91 μmol/mol creatinine) were both higher than those of Japanese smokers (0.26 and 0.12 μmol/mol creatinine) and nonsmokers (0.04 and 0.06 μmol/mol creatinine), respectively. The difference between smokers and nonsmokers was more significant for 2-OHF than for 1-OHP, reflecting the higher intake of fluorene in the vapor phase by the smoking. Moreover, the higher urinary levels of both 2-OHF and 1-OHP were observed in Thai nonsmokers than those in Japanese nonsmokers, suggesting the higher background exposure to PA...

Dioxins are present as impurities in agrochemicals applied to the soil. To examine the possibility that dioxins end up in the atmosphere, we compare the homologue composition and some characteristic isomer distributions of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in air and soil samples collected at sites in Ishikawa Prefecture, Japan. Tetrachloro dibenzo-p-dioxin (TeCDD) was the most abundant PCDD homologue in the air samples, while octachloro dibenzo-p-dioxin (OCDD) was the most abundant PCDD homologue in the soil samples. However, at several site, the most predominant homologue in the soil sample was not OCDD but TeCDD. The mean ratio of TeCDDs to PCDDs in the air samples was significantly smaller in winter than in other seasons, but no such seasonal variation was observed in the distribution of PCDF homologues. The abundance of PCDF homologues was inversely related to the number of chlorine substitutions in the air samples. The contribution of each toxic isomer (2,3,7,8-chlorine-substituted) to the total toxicity equivalency quantity (TEQ) in the air samples tended to be intermediate between their contributions to the TEQs in the soil and cinder samples. This result suggested that the air samples were influenced by both soil/dust suspension and combustion. In order to clarify the contributions of agrochemicals to dioxins in the air, we focused on the ratios of several characteristic isomers that are indicators of agrochemicals (chloronitrophen: CNP and pentachlorophenol: PCP) to their homologues. Significant decreases in the ratios of the isomers to their homologues in the air samples were observed only in winter, probably as a result of suppression of dust suspension by the snow cover. Therefore, the contribution of combustion to the dioxin concentration in the air was thought to be relatively large in winter.

Polycyclic aromatic hydrocarbons (PAHs) were analyzed in maternal blood and fetuses from Fischer 344 rats exposed to diesel exhaust (DE) during pregnancy, and in breast milk from rats exposed to DE during pregnancy and lactation using high performance liquid chromatography with fluorescence detection. Concentrations of phenanthrene (Phe), anthracene (Ant) and benz[a]anthracene (BaA) were significantly higher in maternal blood of the DE group than those of the control group. Concentration of Phe in fetuses of the DE group was significantly higher than those of the control group. Concentrations of fluorene, Ant, fluoranthene (Flu), pyrene (Pyr), BaA and chrysene (Chr) tended to be higher in fetuses of the DE group. The levels of Ant, Flu, Pyr and Chr in breast milk from the DE group were significantly higher than those of the control group. These results indicate that PAHs taken into mother rat by the inhalation of DE are transferred into fetuses via placenta and into breast milk. This is the first report to clarify the transportation of inhaled PAHs into fetuses and breast milk from mother rats.

An automatic HPLC system for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs, nitroarenes) in airborne particulates was previously described (Anal. Chim. Acta, 2001, 445, 20). Some problems with this system were that it generated a peak originating from an ascorbic acid solution that elutes at a retention time close to that of 1,6-dinitropyrene (DNP), and that it was able to analyze only 1,3-, 1,6-, 1,8-DNPs and 1-nitropyrene (1-NP). Here, we describe an improved system that effectively removes the interfering peak by introducing an ODS column just after the pump for the ascorbic acid solution, and which is capable of analyzing several additional compounds (2-, 4-NPs, 2-nitrofluorene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitroperylene and 6-nitrobenzo[a]pyrene etc.). ). The improved sensitivities were achieved by concentrating the compounds in a benzene-ethanol extract from airborne particulates, by increasing the loading time of the sample solution from 20 to 38 min, and by increasing the flow rate of an ascorbic acid solution from 1.3 to 1.8 mL/min.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair. Fifteen kinds of PAHs classified as priority pollutants by the US EPA were quantified with four perdeuterated PAHs as internal standards. After 50 mg hair samples were washed with n-hexane to remove external contamination of PAHs, the samples were digested in 2.5 M sodium hydroxide. The digests were extracted with n-hexane and then analyzed by HPLC. Eleven kinds of PAHs were identified in hair samples of 20 subjects, and 10 kinds of PAHs were eventually quantified using the internal standards. For anthracene, chrysene and benzo[k]fluoranthene, significant differences were observed between smokers and non-smokers. Although benzo[b]fluoranthene, dibenz[a,h]anthracene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were observed in the particulates of indoor and outdoor air, they were not detected in all hair samples. The analysis of PAHs in human hair should be useful as a new biomarker to evaluate the exposure to PAHs.

乾期 (2002年1月～2月) と雨期 (2001年8月) にバンコク市内4個所 (ジャンカセン, ディンデン, チャオプラヤおよびラブラナ) で, ハイボリューム・エアーサンプラーによってエアロゾルを24時間捕集し, エアロゾル組成を求めた.乾期のTSP, PM10, SO2およびNO2濃度は雨期の2倍ほど高かった.これは, 雨期に粒子状およびガス状物質がwash-outによって除去されるためと思われた.水溶性イオンの中ではCa2+とNH4+濃度が雨期に比べると顕著に高かった.NO3-とSO42-濃度は良好な相関性 (R=0.85) を示した.エアロゾルの酸性化を支配するNO3/nss-SO4は雨期に0.37で乾期には0.69となった.Cl/Na (当量比) は雨期に主風向の影響を受けて海塩粒子組成 (1.18) に接近した.1月19日と25日には, 市内の大渋滞によって視呈が著しく低下し, これに対応してNO3-とSO42-濃度が増大した.その後, 高濃度のC2O42-が検出された.TSPはFe, Mn, Ca2+およびK+と良好な相関性を示した.重金属の中ではAlとFe濃度が高く, Pb濃度はWHOの長期基準 (0.5～1.0μg/m3) 未満であった.付近に大規模な重油燃焼系の発生源がないのでVとNi濃度は低かった.分析元素の濃縮係数 (EF) を求めるとZn, Pb, NO3-N, SO4-SおよびTC (全炭素) のEFが大きく人為寄与が疑われ, Fe, Mn, Al等は自然寄与と思われた.なお, 交通量の多いディンデンでは, TSPや重金属濃度の他に, PAHやTCが高くなる特徴が認められた.

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of thiobencarb sulfoxide, which is produced by S-oxygenation of thiobencarb, a herbicide, using γ-cyclodextrin together with sodium dodecyl sulfate. Factors affecting chiral resolution and migration time of thiobencarb sulfoxide were studied. The optimum running conditions were found to be 20 mM phosphate-5 mM borate buffer (pH 8.5) containing 60 mM hydroxypropyl-γ-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of +20 kV at 20°C using direct detection at 220 nm. Under these conditions, the resolution (Rs) of racemic thiobencarb sulfoxide was approximately 1.7. Thiobencarb was treated with a rat liver microsomal fraction containing cofactors (known as S9mix). The ratio between (+)- and (-)-thiobencarb sulfoxide was found to be 15:85. It was also found that the ratio between (+)-and (-)-thiobencarb sulfoxide produced in soil spiked with thiobencarb was 3:7. These results indicated marked enantioselectivities for these metabolisms. The activities of thiobencarb, (+)- and (-)-thiobencarb sulfoxides on 5α-dihydrotestosterone-and 17β-estradiol-induced transcriptions were also investigated. Whereas thiobencarb and (+)-thiobencarb sulfoxide did not show any activities, (-)-thiobencarb sulfoxide showed significant anti-estrogenic and anti-androgenic activities, suggesting that thiobencarb sulfoxide can act as both an enantioselective anti-estrogen and an enantioselective anti-androgen.

In this study, airborne particulates were collected at three sites, two in a downtown area and the other in a suburban area of Kanazawa, Japan in each season for 7 years. Two polycyclic aromatic hydrocarbons (PAHs), pyrene (Py) and benzo[a]pyrene (BaP) and four nitropolycyclic aromatic hydrocarbons (NPAHs), 1-nitropyrene (NP) and 1,3-, 1,6-, and 1,8-dinitropyrenes (DNP) were determined by high-performance liquid chromatography with fluorescence and chemiluminescence detection. At the downtown sites, the mean concentration of each DNP was about two orders of magnitude lower than that of 1-NP and more than three orders of magnitude lower than those of Py and BaP. This tendency reflected the composition of PAHs and NPAHs in diesel-engine exhaust particulates. Concentrations of these PAHs and NPAHs were higher at the downtown sites than at the suburban site, suggesting the dilution of these compounds during the transportation from the downtown to the suburban area. The concentration ratios of NPAHs to PAHs were larger at the downtown sites than at the suburban site. Studies using UV light and sunlight showed that degradation of NPAHs was faster than that of PAHs. Thus, the lower concentrations of NPAHs in the suburban sites may be due to their being photodegraded faster than PAHs during the atmospheric transportation from the downtown area to the suburban area.

A high-performance liquid chromatographic method has been developed for the quantification of 1-hydroxypyrene (1-OHP) in human urine using deuterated 1-hydroxypyrene ([2H9]1-OHP) as an internal standard with fluorescence detection. [2H9]1-OHP was prepared enzymatically from deuterated pyrene ([2H10]Pyr) with cytochrome P450 1A1. It eluted immediately prior to non-deuterated 1-OHP on alkylamide-type reversed-phase columns and had nearly the same fluorescence characteristics as non-deuterated 1-OHP. The detection limit was 0.1 microg/L and the calibration range was from 1 to 100 nmol/L. Urine sample treatment involved enzymatic hydrolysis followed by solid-phase extraction using Sep-Pak C18 cartridges. The proposed method was used to determine urinary 1-OHP in smokers and non-smokers.

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of vinclozolin, which has been used as a fungicide and has an anti-androgenic activity, using gamma-cyclodextrin together with sodium dodecyl sulfate. Factors affecting the chiral resolution and migration time of vinclozolin were studied. The optimum running conditions were found to be 20 mM phosphate-5 mM borate buffer (pH 8.5) containing 50 mM gamma-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of 20 kV at 20 degrees C using direct detection at 203 nm. Under these conditions, the resolution (Rs) of racemic vinclozolin was approximately 2.1. The sample was concentrated by solid-phase extraction and was fractionated by HPLC. The peak area ratio of (+)- and (-)-vinclozolins in wine was found to be 2:3, namely, not racemic, suggesting that degradation rates were different between (+)- and (-)-vinclozolins. The anti-androgenic activities of (+)- and (-)-vinclozolins on dihydrotestosterone-induced transcription were also investigated. The anti-androgenic activity of (+)-vinclozolin tended to be stronger than that of (-)-vinclozolin, suggesting the possibility that vinclozolin can act as an enantioselective anti-androgen.

Airborne particulates were collected at a site near a group of factories in Kitakyushu, one of the most industrialized cities in Japan, and at downtown sites in Sapporo and Tokyo, typical large commercial cities. We determined the levels of polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs), which are known to be carcinogenic and/or mutagenic. The atmospheric concentrations of most PAHs with 4-, 5-, and 6-rings were higher in Kitakyushu and Tokyo than in Sapporo. On the other hand, atmospheric concentrations of the strongly mutagenic NPAHs were the highest in Sapporo, intermediate in Tokyo, and lowest in Kitakyushu. The atmospheric concentrations of 1-nitropyrene (1-NP) in Kitakyushu were one order of magnitude lower than those in the two commercial cities. Therefore the concentration ratios of NPAHs to their nucleus PAHs were significantly smaller in Kitakyushu than in the two commercial cities. This result suggested that the atmospheric levels of NPAHs in Kitakyushu were comparatively low, although the air in Kitakyushu was as heavily polluted with PAHs as the air in Tokyo. The concentration ratio of dinitropyrenes (DNPs) to 1-NP in Kitakyushu was much higher than that in the other two cities. A possible reason for the above two differences between the two types of cities is the contribution of chimney exhaust of steel manufacturing plants in Kitakyushu, which contains high concentrations of PAHs.

A liquid chromatographic (LC) method has been developed for the separation of 11 monohydroxybenzo[a]pyrenes (OH BaPs) positional isomers, and for their detection using electrospray ionization mass spectrometry (ESI-MS). All OH BaPs isomers were separated on an octadecylsilyl (C18)-bonded amorphous organosilica column utilizing gradient elution with acetonitrile–water and triethylamine (TEA) at pH 11.0 and determined by MS, except 2- and 8-OH BaPs which were coeluted. The lower detection limits were in the range from 1.6 µg/L for 12-OH BaP to 12 µg/L for 5-OH BaP without any sample enrichment. The relative standard deviations of area response were in the range from 1.8% (9-OH BaP) to 4.9% (12-OH BaP) except for 9.4% (7-OH BaP). The developed method was successfully applied to incubation mixtures of BaP and CYP1A1/epoxide hydrolase. This method identified 1-, 3- and 9-OH BaPs as the major metabolites, and 2- (and/or 8-) and 12-OH BaPs as the minor metabolites in the incubation mixture. Copyright © 2002 John Wiley & Sons, Ltd.

Diesel exhaust particulate matter (DEP) was collected from three commercial diesel engine vehicles and DEP extracts (DEPEs) were prepared from each. The estrogenic and antiestrogenic activities of the DEPE samples were examined using an estrogen response element (ERE)-driven luciferase reporter gene assay in MCF-7 human breast cancer cells. While DEPE samples did not exhibit significant estrogen agonist activity alone, they suppressed 17 g -estradiol (E 2 )-induced luciferase activity without decreasing cell viability. Several polycyclic aromatic hydrocarbons (PAHs) acting as aryl hydrocarbon receptor (AhR) agonists were detected in the DEPEs and their concentrations were higher in DEPE samples with higher antiestrogenic activity. DEPE samples elevated cytochrome P450 (CYP) 1A1 activity. f -Naphthoflavone, an AhR antagonist, and SKF-525A, a nonselective CYP inhibitor, reversed and enhanced the antiestrogenic activity of DEPE samples, respectively. These results demonstrate that DEPE has antiestrogenic act...

Airborne particulates were collected simultaneously at Vladivostok, Kanazawa and Toyama. 1,3-, 1,6-, 1,8-Dinitropyrenes (DNPs), 1-, 2-, 4-nitropyrenes (NPs), 2-nitrofluorene and 6-nitrochrysene in the particulates were analyzed by HPLC with chemiluminescence detection. All compounds were detected not only in Kanazawa but also in Vladivostok and Toyama. Moreover, five unknown peaks which might be nitropolycyclic aromatic hydrocarbons were detected only in chromatograms of Vladivostok. The [DNPs]/[1-NP] ratios at all stations were near the value found in diesel-exhaust particulates, suggesting that one of the main contributors of these compounds was diesel-engine vehicles not only in Kanazawa and Toyama but also in Vladivostok. However, the difference in traffic volume at two stations in Vladivostok did not have a significant effect on the concentrations of DNPs and NPs in contrast to two stations in Kanazawa. These results suggested sources other than diesel-engine vehicles also contributing to DNPs and NPs in Vladivostok. Seasonal variations similar to those in Kanazawa were observed in Vladivostok and Toyama. As in Kanazawa, the [2-NP]/[1-NP] ratio in Vladivostok was larger at the lighter traffic station than that at heavier traffic station. This suggested that, as in Kanazawa, 2-NP was formed in the atmosphere in Vladivostok.

A HPLC system with on-line reducer, clean-up and concentrator columns has been developed for determining nitropolycyclic aromatic hydrocarbons (NPAHs). Maximum efficiency of the reducer column (Pt/Rh-coated alumina) was achieved after the column was activated with a flow of air/hydrogen at 350°C for 3 h and when ethanol–acetate buffer was used as the sample carrier solution. After a benzene–ethanol extract from airborne particulates was washed with sodium hydroxide, sulfuric acid and water, an aliquot was injected into the system. NPAHs were separated from interfering substances by the clean-up column and reduced to their amino derivatives by the reducer column. The derivatives were concentrated on the concentrator column and eluted into the separator column. NPAHs such as 1,3-, 1,6-, 1,8-dinitropyrenes and 1-nitropyrene were determined chemilumigenically with linear calibration graphs from 1×10−11 to 1×10−8 and 1×10−10 to 1×10−7 M, respectively, at an injection volume of 100 μl. These NPAHs in airborne particulates were determined by the proposed method.

In this study, we performed fractional partition and determination of metals in river water by filtering the sample through a membrane filter with pore size of 0.22 µm immediately after sampling. We considered that the total amount of metal was contained in the nonfiltered sample, and fractional determination was performed separately using atomic absorption spectrometry of the metal in both the filtered and nonfiltered samples. The results showed a different pattern of aluminum distribution compared with other elements.

Benzo[a]pyrene (BaP), a major environmental pollutant, is metabolized in vivo and produces many hydroxy derivatives. The estrogenic/antiestrogenic activities of twelve monohydroxy derivatives of BaP (1- through 12-OH species) were investigated using competition binding to human estrogen receptor (hER)α and hERβ, and the gene expression assay of the yeast two-hybrid system. BaP and 5-OH BaP did not bind to either hER. The other monohydroxy derivatives bound to both hERs. These compounds bound more strongly to hERβ than to hERα. Using the yeast two-hybrid assay system, 1-, 2-, 3-, and 9-OH BaP induced β-galactosidase with hERβ but not with hERα. This suggested that these compounds were estrogenic. In the presence of 10-9 M 17 β-estradiol, 8-OH BaP inhibited the induction of β-galactosidase. Because 8-OH BaP did not affect cell growth, it appeared to be an antiestrogen. The present study shows that most of the monohydroxy derivatives of BaP bind to estrogen receptors (ERs), and several of them have estrogenic or antiestrogenic activity.

Airborne particulates were collected during the same periods in downtown Kanazawa, Sapporo and Tokyo and polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) were determined together with several heavy metal elements and water-soluble inorganic ions. The mean concentrations of metal elements and inorganic ions were the highest in Tokyo, followed by Sapporo and the lowest in Kanazawa both in winter and summer, and were strongly dependent on the amount of airborne particulates. These tendencies were different from the cases of PAHs and NPAHs. Correlations between the organic compounds (PAHs and NPAHs) and several inorganic pollutants were strong only in winter samples of Sapporo, but not so strong in Kanazawa and Tokyo. Because PAHs and NPAHs and several combustion-origin inorganic substances are, in general, detected mainly in the fine airborne particulates, it was reasonable that the significant correlations were found only in the winter samples of Sapporo where the generation of the coarse and crustal particulates were suppressed by snow or ice. Though the atmospheric concentrations of PAHs and NPAHs in Kanazawa were much lower than in Sapporo, the correlation among the NPAH compounds and between PAHs and NPAHs were surprisingly similar in these two cities. The strong correlations indicated that the atmospheric behaviors of the PAHs and NPAHs studied were similar in the two cities and that the source of PAHs and NPAHs might be the same. On the contrary, strong correlations were not observed in Tokyo, suggesting the possibility of multiple sources or formations/degradations of PAHs and

An oil spill accident happened to a Russian tanker Nakhodka with a cargo of heavy oil type C in the Sea of Japan on January, 1997 and the spilled oil severely polluted the coast of Japan and damaged the environment. In this study, androgenic and antiandrogenic activities of C-heavy oil crude extracts prepared with ethanol were evaluated on two androgen-responsive cell lines, a Shionogi mouse mammary carcinoma SC115 and a human prostate carcinoma LNCaP. Oils used in this study were the Nakhodka and a commercial C-heavy oils. Assessment of androgenic and antiandrogenic activities was made on the basis of effect on proliferation (SC115) and prostate specific antigen (PSA) production (LNCaP cells) in the absence and the presence of 0.5 nM dihydrotestosterone (DHT). While both extracts exerted almost no effect on the proliferation and PSA production of SC115 and LNCaP cells in the absence of DHT, the extracts significantly inhibited the DHT-induced proliferation and PSA production of the cells in the presence of DHT, indicating that the C-heavy oils contains antiandrogenic compounds. It has been known that androgen receptor (AR) expressed in LNCaP cells has mutation in ligand-binding domain and consequently, its transcription promoting action after ligand-binding is different from that of normal AR. Cyproterone acetate (CA), an androgen antagonist, and 17β-estradiol, an estrogen, stimulated PSA production and their stimulatory effects were additive to that of DHT in LNCaP cells, while CA inhibited DHT-induced proliferation and 17β-estradiol showed quite weak agonistic effect in SC115 cells. Therefore, a part of antiandrogenic effects of the oil extracts was considered to be mediated through mechanism other than direct transcriptional activation by activated AR. Then, a few polycyclic aromatic hydrocarbons (PAHs) that are considered not to bind to AR were examined for their androgenic and antiandrogenic effect. Benzo[ a ]anthracene (BaA), benzo[ k ]fluoranthene (BkF) and benzo[ a ]pyrene (BaP) suppressed DHT-induced proliferation and PSA production of SC115 and LNCaP cells in a concentration-dependent manner. The antiandrogenic effect of the two heavy oil extracts was considered to be due in part to PAHs.

Two bacterial strains, N-21 and N-22, which were isolated from natural oil-producing wells at Kurokawa, Niigata Prefecture, Japan, were identified as Pseudomonas cepacia by using the phylogenetic analysis based on the 16SrDNA sequences. Both strains were gram negative rods capable of decomposing naphthalene. The degradation rates were 2.24 × 10-5 M/h in both strains. The main metabolite was cis-1,2-dihydro-1,2-naphthalenediol. Salicylaldehyde, salicylic acid, gentistic acid and catechol were also identified. These metabolites were less mutagenic than naphthalene itself in the Ames test using the S. typhimurium TA100 strain. The mutagenicity of naphthalene also decreased by the two bacterial strains.

Purpose: The authors have previously reported that trans-bis(n-valerato)(1R,2R-cyclohexanediamine) (oxalato)platinum(IV) (C5-OHP), an oxaliplatin derivative, is an orally active antitumor agent in an intraperitoneal (i.p.) L1210 murine leukemia model. In this study, several oxaliplatin derivatives of the general formula trans-(carboxylato)chloro(1R,2R-cyclohexanediamine)(oxalato)platinum(IV) were synthesized in order to find new derivatives with greater oral activity than C5-OHP in a clinically predictive tumor model. In the formula, the carboxylate and chloride ligands are situated in axial positions. Methods: Four complexes with the axial carboxylate ligands n-butyrate, n-valerate, n-caproate or n-heptanoate were synthesized and designated C4-OHP-Cl, C5-OHP-Cl, C6-OHP-Cl and C7- OHP-Cl, respectively. The oral antitumor activity of the complexes was evaluated against the murine reticulosarcoma M5076 implanted subcutaneoulsy (s.c.) in to male BDF1 mice. The complexes were administered orally daily for 5 days in two cycles initiated on days 5 and 12 postimplantation. The physicochemical properties were examined by measuring the concentrations of the complexes in test solutions at intervals by HPLC. The pharmacokinetic behaviors of C5-OHP-Cl, C6-OHP-Cl and C5-OHP following a single oral administration were studied in non-tumor-bearing male BDF1 mice. Results: Of the complexes synthesized in this study, C5-OHP-Cl, which exhibited high activity in the i.p. L1210 model, was found to be orally active in the s.c. M5076 model while C5-OHP was not. The in vitro reduction of the complexes by ascorbate was much more rapid than that of C5-OHP, while the complexes were more stable than C5-OHP in HCl-acidic and alkaline solutions. Pharmacokinetic study showed that Cmax and AUC0–24h values of plasma total and filterable platinum of C5-OHP-Cl were four to six times greater than those of C5-OHP, indicating that C5-OHP-Cl was absorbed more than C5-OHP. Conclusion: C5-OHP-Cl was found to be a superior l-OHP derivative C5-OHP, exhibiting significant oral antitumor activity in the s.c. M5076 model. The enhanced activity of C5-OHP-Cl was considered to be due in part to increased susceptibility to reduction and increased gastrointestinal absorption. C5-OHP-Cl is a suitable candidate for further study as an oral cancer chemotherapy agent.

エストロゲン作用の新しいin vitroアッセイ法として,ヒト乳がん由来MCF-7細胞におけるプロゲステロン受容体(PgR)の発現量を指標とする方法を提案し,現在最もはん用されているE-Screenアッセイ法と比較するとともに,C重油のエストロゲン作用を検討した.試料としては,重油のエタノール抽出物を用いた.PgRの発現量をウエスタンブロット法で測定したところ,17β-エストラジオール(E2)処理によりPgRの発現量が高まり,更にE2アンタゴニストであるタモキシフェンの処理を加えるとPgRの発現量は減少した.E-Screenアッセイにおいても,E2及びタモキシフェン処理に対して類似の応答を示し,PgR発現量がエストロゲン作用の良い指標であることが明らかになった.次に,重油抽出物のエストロゲン作用について検討した.E2非存在下では重油抽出物の処理でわずかながらPgRの発現量が高まったが,E2存在下ではPgRの発現量は減少した.以上の結果から,重油中には弱いエストロゲン作用を示し,E2アンタゴニストとして作用する化合物が含まれることが明らかとなった.また,重油抽出物の処理では死細胞数の増加が観察された.重油や環境試料のように細胞死を引き起こす成分を含む可能性がある試料については,本研究のPgRの発現量を指標とする方法が適していると考えられた.

金沢大学理工研究域自然システム学系, Black and white hair samples were obtained from black-, gray-(i. e., a mixture of black and white hair) and white-haired methamphetamine (MA) users, and MA and amphetamine (AP) were determined by HPLC using chemiluminescence detection (CL-HPLC). MA and AP were detected in black hair, and were found in the part of the hair that grew during the period of MA use. In the same subjects, MA concentrations were lower in white hair than in black hair and AP was not detected in white hair. This difference may be related to the affinity of MA and AP for melanin.

Trans-bis(n-valerato)(1R,2R-cyclohexanediamine)(oxalato++ +)platinum(IV) (C5-OHP) is an orally active platinum complex we prepared. The gastrointestinal absorption of C5-OHP was examined in rats and compared with those of C5-OHP analogs which have a general formula of trans-bis(n-OCOCnH2n+1)(1R,2R-cyclohexanediamine)(oxalato )platinum(IV) as well as C5-OHP. The complexes did not show significant differences in pharmacokinetic behavior after i.v. injection. Plasma platinum level after a single oral administration at a dose was higher for a complex with higher water solubility. The intestinal absorption rate measured by an in situ recirculating perfusion technique was higher for a complex with higher lipophilicity. These results indicate that the water solubility is a more dominant factor than the lipophilicity in the gastrointestinal absorption of the complexes. Then, the effects of surfactants and alpha-cyclodextrin (alpha-CD) on the solubility of C5-OHP was studied. Among the agents tested, alpha-CD showed the highest effect in increasing the solubility. Administration of C5-OHP together with alpha-CD gave approximately three times higher plasma platinum levels than administration of C5-OHP alone. Water solubility was found to be a dominant factor in the gastrointestinal absorption of C5-OHP and its analogs.

The Russian tanker Nakhodka met a hull-broken accident in sailing in the Sea of Japan on January 2, 1997, releasing approximately 6200 kl of heavy oil into the Sea of Japan. The spilled oil was driven by the prevailing winds and water currents and polluted the coastline from the Shimane Prefecture through the Yamagata Prefecture. While much effort was made to remove the reached oil, many workers engaged in oil-removing complained of an eyeache, giddiness, a headache, etc. These symptoms were considered to be caused by volatile chemicals in the oil. Although the greater part of the oiled coastline was cleaned shortly after the oil pollution, there is concern that sands, rocks and sea water of the shore were still contaminated with chemicals included in the oil. Furthermore, some part has remained not to be treated. Extensive scientific studies were conducted to 1) evaluate the influence of volatile chemicals in the spilled oil on the health of oil-removing workers, 2) assess the impact of the spill on the ecosystem, 3) where and how long the contamination by the oil or chemicals persists in the environment. This paper reviews the activities and researches made after the Nakhodka oil spill accident.