Heme is believed to control expression of genes involved in the synthesis of globins and heme itself. However, heme-regulated transcription factor had not been identified in vertebrates. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. Recently, we found that heme binds specifically to Bach1 and regulates its DNA binding activity. Deletion studies demonstrated that the heme binding region of Bach1 is confined within its C-terminal region that possesses four di-peptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased level of intracellular heme inactivates the repressor Bach1, resulting in induction of a host of genes with MAREs.

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Trans fatty acids have several beneficial aspects for processed foods owing to their characteristic structures. These very characteristic structures, in turn, have been suspected to be associated with the possibility that trans fatty acids affect the development of several health problems, including coronary heart disease, and fetal and infant neurodevelopment and growth, and childhood allergies.

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Amphetamines and their analogues have undergone a cycle of popularity as recreational drugs in Japan. The current wave of popularity began in the early 1990s and spread throughout the country. More recently, not only 3,4-methylenedioxymethamphetamine (MDMA) but also other designer amphetamines analogues, including p-methoxyamphetamine (PMA), 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), etc., have been extensively and increasingly abused, mainly among juveniles. This minireview presents an update on the amphetamines and their analogues by focusing on clandestine tablets encountered in Japan recently.

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Effect of oxidative stress induced by vitamin E-deficiency and/or nitrogen dioxide (NO₂) inhalation on Type IV and Type I allergy responses of mice was investigated. Mice were fed a vitamin E-adequate diet (control: C group) or a vitamin E-deficient diet (-E group). The vitamin E content in the blood of C and -E groups was 1.58 and 0.3 μg/ml, respectively. Mice of the C and -E groups were exposed to air or air containing NO₂ (5-6 ppm) (C + NO₂ and -E + NO₂ groups) by feeding them the corresponding diets for 1-2 week. In the sensitization with 2,4-dinitrochlorobenzene (DNCB), the degree of lymph node cell proliferation of mice of the C + NO₂ group, as assessed by lymph node assay, was similar to that of mice of the C group. While the degree of cell proliferation of mice of the -E group was lower than that of mice of the C group, the degree of cell proliferation of mice of the -E + NO₂ group was higher than that of mice of the C and C + NO₂ groups. While the DNCB-sensitized IgE levels of mice of the C + NO₂ group were similar to those of mice of the C group, the IgE levels of mice of the -E and -E + NO₂ groups were higher than those of the C and C + NO₂ groups. While the trimellitic anhydride (TMA)-sensitized IgE levels of mice of the -E group were similar to those of mice of the C group, the IgE levels of mice of the C + NO₂ and -E + NO₂ groups were much higher. These results suggest that allergen-sensitized type IV and I allergy responses of mice are enhanced by oxidative stress induced by vitamin E-deficiency and/or NO₂ inhalation.

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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.

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Electron-beam irradiation facilities are commercially utilized for food irradiation. This study investigates whether dosimeters used to detect gamma-rays can also be used to accurately detect electron-beam irradiation. The irradiation souses for gamma-ray at the Takasaki Institute were used throughout this study. Four kinds of dosimeters, RadiaChromic (RC), GammaChromic (GC), Amber3042 (AM), Radix (RX), and alanine (AL) were irradiated at doses from 0.1 to 60 kGy using a 5 MeV electron-beam (EB) at the Japan Irradiation Service Co. Ltd. (Japan). AL was submitted to the National Physics Laboratory in order to evaluate the doses that AL were absorbed. At low-level dose range, all dosimeters indicated correct values. This means those dosimeters were usable to accurately detect electron beam irradiation. But at higher range they all required correction due to either temperature effects or varying sensitivity to electron-beam exposure. Dose-depth profiles were obtained using ham and cheese samples, in order to check penetration of EB by the calibrated machine.

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Cyclooxygenase (COX), which catalyzes the synthesis of prostaglandins from arachidonic acid, has two isoforms; COX-1 and COX-2. A large body of evidence exists to suggest that COX-2 is important in gastrointestinal cancer. In order to determine whether COX-2 is expressed in transitional cell carcinoma (TCC) of the human bladder as well as in gastrointestinal cancer, we investigated COX-2 expression in human TCC by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical analysis, and we found that normal bladder epithelium did not express COX-2 and that COX-2 was markedly up-regulated in human bladder TCC. In nontumor tissues, COX-2 immunostaining signals were observed only in lymphoid follicles. Furthermore, the intensity and extent of COX-2 immunostaining in the bladder cancer tissues were scored and the relationship to tumor grade and stage was investigated. The levels of COX-2 expression were correlated with the tumor grade; from grades 1 to 3, there was a stepwise increase in the COX-2 immunostaining score. These findings suggested that an increase in COX-2 expression may be associated with bladder carcinogenesis as well as gastrointestinal carcinogenesis, and that it may be useful as a biomarker in bladder cancer.

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As a sustained release preparation of diclofenac sodium (DF-Na), we developed a preparation (SR318B) that was expected to exhibit a persistent analgesic effect by once-daily oral administration. In this study, we investigated the persistence of the analgesic effect of SR318B using female cynomolgus monkeys with arthritis induced by sensitization with bovine type II collagen combined with FreundÕs complete adjuvant. Cynomolgus monkeys in which arthritis was induced to the same severity received oral administration of SR318B (1 mg DF-Na/kg) once daily for 14 days. The mean ellipsoid area of the proximal interphalangeal joints in the monkeys slightly increased before administration on day 3 in the control group. In contrast, the area decreased after day 3 and significantly decreased on day 13 in the SR318B treatment group. The plasma diclofenac (DF) level reached the highest point at 6 hr after administration on day 13, then decreased. The plasma level was lower than the quantification limit 20 hr after administration on both days 0 and 13. In contrast, DF was detected in synovial fluid 24 hr after the initial and final administration. Based on the above findings, SR318B exhibits a persistent analgesic effect on collagen-induced arthritis. While the plasma DF concentration decreased to a level lower than the quantification limit at 20 hr, DF was still detected in the synovial fluid 24 hr after administration. Therefore, the retention of DF in inflammatory regions may play an important role in the analgesic effect.

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Cadmium is a unique heavy metal that stimulates the secretion of plasminogen activator inhibitor type 1 (PAI-1) from vascular endothelial cells. However, it has been incompletely understood whether cadmium stimulation of PAI-1 secretion actually results in a reduction of endothelial fibrinolytic activity and whether the stimulation results from an induction of endothelial PAI-1 synthesis. To address these questions, human umbilical vein endothelial cells were cultured with cadmium chloride in the presence or absence of actinomycin D, H-7 or HA1004. The activity of tissue type and urokinase type plasminogen activators (t-PA and u-PA, respectively) in the conditioned medium was analyzed by fibrin zymography and mRNAs coding t-PA, u-PA and PAI-1 were determined by quantitative reverse transcription-polymerase chain reaction. Results of the experiments indicate that cadmium reduces the activity of both t-PA and u-PA in vascular endothelial cells through induction of PAI-1 synthesis which is mediated by protein kinase C activation. Since the PAI-1 induction by cadmium was observed in neither human vascular smooth muscle cells nor human fibroblasts, it was suggested that vascular endothelial cells are a particular cell type of which PAI-1 synthesis is stimulated by cadmium.

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Selenium-binding protein (SeBP) is one of the cytosolic proteins which is believed to bind to selenium without containing selenocysteine. Although its physiological role remains to be identified, it has recently attracted interest due to its participation in the late stages of intra-Golgi protein transport. In a previous study, we found that SeBP in rat liver is significantly increased following administration of aryl hydrocarbon (Ah)-receptor ligands, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and 3-methylcholanthrene (MC). Little research has been carried out to examine the properties of the inducibility of SeBP by xenobiotics. The objectives of this study were to analyze the control of transcription of the message by Ah-receptor ligands. In this report, we cloned an inducible rat SeBP. The resulting PCR product consisted of the full coding sequence for the 472 amino acids. The deduced amino acid sequence exhibited homologies with mouse SeBP, mouse acetaminophen-binding protein and human SeBP of 92%, 93% and 87%, respectively. We also measured the mRNA level by quantitative reverse transcriptional (RT)-PCR. Treatment of rats with PCB126 resulted in significant induction of the mRNA of this protein in the liver compared with the control level. Our data represent the first report showing that SeBP is induced by an Ah-receptor ligand at the mRNA level.

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We investigated the functional significance of peripherally distributed glutamate, including N-methyl-D-aspartate (NMDA), receptors in humans. Magnesium sulfate (MG) and ketamine hydrochloride (KET), NMDA receptor antagonists, were injected into the skin of the medial region of forearm of healthy volunteers to investigate whether these substances are capable of changing the sensory transmission. We found that intracutaneous injections of MG and KET cause hypesthesia to noxious and innoxious mechanical stimulations. These results suggest that peripheral NMDA receptors are involved not only in sensory acceptance of noxious mechanical stimulation, but also in transmission of innoxious mechanical stimuli. The observation that the state of NMDA receptors in the skin may be affected by intracutaneous injection of their antagonists lays ground for possible clinical applications.

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A mutagenicity test was conducted for 1,4-pentadiene-3-ol, 2,4-hexadiene, isoprene, cis- and trans-piperylene in the bacterial mutation assay using Salmonella typhimurium (S. typhimurium) strain TA 100, TA98, TA1535 and Escherichia coli (E. coli) WP2uvrA/pKM101 with and without metabolic activation by S9 mix in the preincubation method. The mutagenicity of 1,3-butadiene was tested by the gas exposure method. Mutagenicity was weakly positive for 1,4-pentadiene-3-ol in TA 100 with S9 mix and was pseudopositive for WP2uvrA/pKM101 without S9 mix. Mutagenicity was very clear for 1,3-butadiene in 10 fold concentrated TA1535 with S9 mix. The 2,4-hexadiene, isoprene, cis-piperlylene and trans-piperlylene were not mutagenic on S. typhimurium TA98, TA100, TA1535 or E. coli WP2uvrA/pKM101 with or without metabolic activation. It can be concluded from these results, that chemicals, containing double bond carbon (C=C) chemical generally tend to show weak mutagenicity in the presence of the metabolic activation system.

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Ortho-tyrosine detection method is used for the detection of irradiated protein-rich foods. A new procedure determining o-tyrosine content was examined in regard to the identification of food having undergone ionization treatment. A new fluorometric HPLC method allows the detection of irradiated foods at 10 kGy. This method was compared with an electron spin resonance (ESR) method recommended by European countries for the detection of irradiated foods. The method is applied only those food samples that contain bone or cellulose. The dose response of o-tyrosine production was linearly increased upto 30 kGy. Chicken bones were dried in a desiccator, after which ESR signals were recorded in the samples. The dose response of the ESR signal was observed two weeks after irradiation, and the equation for curve fitting was a quadratic polynominal. The ESR signal intensity correlated with the logarithm of o-tyrosine production, a close relationship observed.

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We developed a yeast two-hybrid assay using a human-type estrogen receptor (hERα) to screen test chemicals reporting weak estrogenic activity at high concentrations. As a result of exposure to several test chemicals, viable yeast cells showed a decrease in number compared with a control. It was shown that this phenomenon was due to fungicidal effects of the test chemicals within the yeast cell or to inhibition of yeast cell proliferation. Therefore, in performing a yeast two-hybrid assay, it is necessary to monitor yeast cell proliferation by methods such as the measurement of the number of viable cells, and it is necessary to screen test chemicals within a concentration range where these chemicals show neither fungicidal effects towards the yeast cell nor inhibition of yeast cell proliferation.

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The efficacy of two antimicrobial preservatives, 2-phenoxyethanol (2-PE) and Thimerosal, was compared in adsorbed diphtheria-purified pertussis-tetanus combined vaccine (DPT vaccine). Thimerosal had strong microbicidal activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi at 25°C and 4°C. 2-PE also had microbicidal activity against these microorganisms, only had microbistatic activity against fungi at 4°C. Neither 2-PE nor Thimerosal affected the potency or toxicity of the DPT vaccine. These data suggest that 2-PE has weaker antimicrobial activity than Thimerosal against yeast and fungi in DPT vaccine at low temperatures.

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We observed and evaluated the feeding behavior of the free-living nematode Caenorhabditis elegans (C. elegans) after exposure to bisphenol A (BPA) and nonylphenol (NP). Exposed organisms were transferred to chemical-free culture medium and their attainment levels (the number of worms reaching the food source divided by the total number of worms on the Petri plate) were recorded after 2, 4, 6, 8, and 24 hr. Results showed a significant decrease in the attainment level of worms exposed to 10 μM and 0.1 μM BPA. However, there was a slight increase in the attainment level of nematodes treated with 1 μM NP. These results differ from previous studies showing NP as being more lethal to nematodes than BPA.

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