This review summarizes technical and administrative improvements in forensic hair analysis for methamphetamine (MA) and designer drugs, recently developed by the present authors' team. These improvements include the establishment of three types of screening methods, and a column-switching liquid chromatographic-mass spectrometric (LC-MS) method for determining such drugs and their metabolites to serve as an alternative confirmation technique. The screening methods include ion mobility spectrometry-based, enzyme multiplied immunoassay technique (EMIT)-based, and immunochromatography-based methods. The column-switching LC-MS method proved to be effective in clearly determining MA and its metabolites, amphetamine and p-hydroxy-MA, which provides indisputable proof of MA intake. Quality assurance measures in forensic hair analysis, such as semiquantitative cross-checkings between the results of different analyses, are also discussed.

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Polychlorinated biphenyls (PCBs) and dioxins have polluted the environment for about 50 years and parts of them have transferred to human being through food chain. Humans are already contaminated with chlorinated compounds at relatively high levels. Yusho PCB poisoning occurred at Northern Kyushu in 1968 and the patients have been suffering from various symptoms for 35 years. High concentrations of toxic PCBs and polychlorinated dibenzofurans in the patients have been very gradually decreased to the levels of only 2 to 6 times higher than those of normal persons. Typical Yusho symptoms of acneiform eruption, dermal pigmentation and increased eye discharge were very gradually recovered with lapse of several years. However, enzyme and/or hormone-mediated signs of high serum triglyceride, high serum thyroxin, immunoglobulin disorder and others are persistently maintained for more than 30 years. Recent tolerable daily intake of dioxins were determined from their hormone-like activities, such as decreased sperm count, immune suppression, increased genital malformation and neurobehavioral effects in the offspring of animals. PCBs are important factors on considering dioxin toxicities to human for the reason that half the dioxin toxicity in normal persons consists of the toxicity of coplanar PCBs. Recent epidemiological studies have reported that higher levels of PCBs and related compounds in the human body associated with various health effects, such as lowering intelligence quotient levels, disorder of thyroid gland, higher rate of endometriosis in women, declining thyroid hormone levels, higher rate of diabetes in pregnant women, lowering age at menarche, and altering play behavior in children at school age.

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Recently, we have reported the chemical properties of some jellyfish proteinaceous toxins. These were the first chemical characterizations of jellyfish protein toxins to be reported. The isolation of the proteinaceous toxins in their active forms was the key step in the studies. We isolated the toxins from three box jellyfish (Cubozoa) species [Carybdea rastoni (C. rastoni), Carybdea alata (C. alata), and Chiropsalmus quadrigatus (C. quadrigatus)]. These toxins showed lethal toxicity to crustaceans and hemolytic activity. Furthermore, the full-length cDNAs and the deduced amino acid sequences of the toxins were clarified. All of these toxins have a molecular weight of around 45 kDa and their amino acid sequences showed homology with each other. The box jellyfish toxins represent a novel bioactive protein family.

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Aggregation and subsequent conformational change of Alzheimer's β-amyloid protein (AβP) enhance its neurotoxicity. Therefore, factors that inhibit or promote conformational changes of AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD). Moreover, recent studies have suggested that a common mechanism is based on the diverse diseases termed "conformational diseases" including neurodegenerative diseases such as AD, prion diseases, Parkinson's disease, and Huntington's disease. These diseases share similarity in the formation of β-sheet containing amyloid fibrils by disease-related proteins and the introduction of apoptotic degeneration. Aluminum, an environmental risk factor for AD, is a widely used cross-linker that causes conformational changes of AβP and other proteins. This report reviews and discusses characteristics of aluminum-induced conformational changes of AβP and their implication in pathogenesis of AD. Taking together our results and those of numerous other studies, we hypothesize that aluminum-induced conformational changes enhance the neurotoxicity of AβP and lead to development of AD.

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The effect of aging on the development of glucose intolerance between 2 kinds of diets, a low-carbohydrate/high-fat (LC/HF) diet and a control diet was investigated. A total of 20 eight-week-old S.D. rats were randomly divided into 2 groups of 10 rats each and at 10, 20, 40, 60 and 80 weeks of age, rats were switched to either control or LC/HF diet for 7 days. At 16:00 on the day following the 7th day of feeding, rats underwent an oral glucose tolerance test (OGTT). LC/HF feeding for 7 days impaired the glucose tolerance in rats. Moreover, insulin secretion decreased with age and insulin resistance increased after 60 weeks of age when rats were fed the LC/HF diet, and fasting plasma free fatty acids (FFA) concentrations increased with age. Thus, we speculated that the age-related impairment of insulin action through the glucose fatty-acid cycle might be closely associated with the onset and development of diabetes mellitus.

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A sensitive and specific double-antibody enzyme immunoassay (EIA) for nociceptin (orphanin FQ)-like immunoreactive substances (nociceptin-IS) was developed. In competitive reactions, the nociceptin-antibody was incubated with both a nociceptin standard (or plasma extract sample) and β-D-galactosidase (β-Gal)-labeled synthetic human nociceptin (delayed addition method). The free and antibody-bound enzyme haptens were separated using an anti-rabbit IgG-coated immunoplate. The enzyme activity on the immunoplate was determined fluorometrically. The present immunoassay allows the detection of 15-700 pg/ml (sensitivity: 1.5 pg, 0.6 pg/well) of nociceptin. Using this EIA, the nociceptin-IS levels in human plasma were determined, and found to be in the range of 5.0 to 16.0 pg/ml. No circadian rhythms in the daytime (9:00-19:00) or effects of eating meals on human plasma nociceptin levels were found. We have established an evaluation system for both nociceptin-IS and substance P-IS levels in 1 ml of human plasma. As for the evaluation of analgesic effects of drugs and pain, this sensitive and specific EIA system for endogenous nociceptin may be valuable for clinical use.

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We previously reported that trans-stilbene is metabolically activated to estrogenic compounds by a liver microsomal enzyme system. In this study, we demonstrated the structural requirement for estrogenic activity of various stilbene derivatives including proestrogens. High estrogenic activity in 4,4′-dihydroxystilbene, 4-amino-4′-hydroxystilbene, 4,4′-dihydroxy-α-methylstilbene, hexestrol, and diethylstilbestrol (DES), moderate activity in 4-hydroxystilbene, 4-aminostilbene, 4-hydroxyazobenzene, and 4-hydroxy-4′-nitrostilbene, low activity in 4-nitrostilbene, 4,4′-dihydroxydibenzyl, resveratrol, and 4-hydroxy-α-methylstyrene, and marginal activity in 4,4′-dimethoxystilbene and 4-hydroxymethylstilbene were observed in an estrogen reporter assay using the estrogen-responsive human breast cancer cell line MCF-7 and a binding assay with rat uterus estrogen receptor. In contrast, α-methylstilbene, 4,4′-dimethoxystilbene, 4-hydroxymethylstilbene, dibenzyl, tolan and azobenzene also exhibited estrogenic activities after incubation with liver microsomes of 3-methylcholanthrene- or phenobarbital-treated rats in the presence of NADPH. These results suggest that the structural requirements for the estrogenic activities of stilbene derivatives are a p-hydroxyl group in the A-phenyl ring, vinyl linkage, and a B-phenyl ring for the maximal activity, and hydrophobicity of the linkage for higher activity as observed in DES. p-Nitro and amino groups in the A-phenyl ring are also effective for the estrogenic activity.

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A Cynomolgus monkey hemodialysis model was used to evaluate the efficacy of a new factor Xa (FXa) inhibitor as an anticoagulant for hemodialysis. We tested the selective FXa inhibitor KFA-1411, using doses of 0.15 mg/kg/hr, 0.3 mg/kg/hr, and 0.6 mg/kg/hr by continuous infusion in combination with a single loading-dose at the start. As a reference control, dalteparin, which is a low-molecular-weight heparin, was used at a dose of 20 IU/kg/hr plus 30 IU/kg single loading. As a monitoring method for KFA-1411, clotting time was measured in blood samples obtained from the body (systemic) and from the extracorporeal circulation (in-circuit). At KFA-1411 doses of 0.3 mg/kg/hr and 0.6 mg/kg/hr hemodialysis was maintained with essentially no change in the intra-bloodline pressure in the extracorporeal circulation. With dalteparin, the pressure increased almost to the upper limit (set at 250 mmHg). The clot-deposition score measured, after testoration of normal circulation, in the dialyzer and air-trap chamber in the extracorporeal circulation was improved in a dose-dependent manner in the KFA-1411 groups. There were no large differences between humans and Cynomolgus monkeys in the in vitro plasma anticoagulant activities of KFA-1411 and dalteparin. These results indicate that this selective FXa inhibitor could be used as an anticoagulant for hemodialysis. Both activated partial thromboplastin time (APTT) and prothrombin time (PT) can be used to monitor the anticoagulant activity in the extracorporeal circulation, and a drug dose that has sufficient anticoagulant efficacy for hemodialysis will prolong in-circuit-APTT about 2 times and in-circuit-PT about 3 times compared to normal.

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The biodegradation capacity of planktonic cells and biofilms obtained from a natural river located in an industrial area was compared through the use of a river die-away biodegradation test. The change in the bacterial community during the biodegradation process was also investigated using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments and multidimensional scaling (MDS) analysis of banding patterns in DGGE gel. The bacterial community structure of biofilm formed on ceramic slides submerged in natural river water were similar to those formed on natural stones in the river bed, that is, ceramic is a more suitable material for forming natural biofilm than glass or polycarbonate. Biodegradation of aniline-derived compounds revealed that aniline was easily biodegraded by both planktonic and biofilm communities. N-methylaniline, a difficult compound to biodegrade, could not be biodegraded by planktonic bacterial cells, although biofilm communities could biodegrade this compound within 7 days. The banding pattern in DGGE gel showed that one dominant bacterium appeared during biodegradation of aniline and the bacterial community was disturbed by the addition of chemicals, although bacteria formed a relatively stable community after biodegradation was completed. DGGE of PCR-amplified 16S rDNA fragments and MDS analysis of banding patterns in DGGE gel are available for monitoring bacterial populations in the biodegradation test system. These results should be useful to improve the river die-away biodegradation test.

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The mutagen 3-nitrobenzanthrone was determined in rainwater. Rainwater was collected at a residential area in Kyoto, Japan. Organic matter containing 3-nitrobenzanthrone in rainwater was extracted using a 3M (St. Paul, MN, U.S.A.) Empore Extraction Disk C₁₈, and the extract was purified on a pyrenylethyl silylated silica gel column. The purified sample was then reduced using a platinum black column heated at 80°C. The reduced product of 3-nitrobenzanthrone, i.e. 3-aminobenzanthrone, was analyzed by normal-phase HPLC with fluorescence detection. 3-Nitrobenzanthrone in the rainwater was determined in the range of 0.07-2.6 ng l^-1. The concentration was higher from May to July and lower from October to December. 1-Nitropyrene was also determined by reversed-phase HPLC with fluorescence detection after platinum black column reduction. 1-Nitropyrene was detected in the range of 0.0056-0.19 ng l^-1, and this concentration range was lower than that of 3-nitrobenzanthrone.

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It has been known that ciprofloxacin augments the humoral and cellular immune systems. The aim of the present study was to determine whether these properties affect the inflammatory response. Adult male rats, ciprofloxacin and pefloxacin were used. The healty rats were treated orally with an equal volumes of ciprofloxacin (50 mg/kg) pefloxacin (50 mg/kg) or distilled water for 10 days. Their effects on the inflammatory response were investigated by testing the formation of formaline-induced edema. The effects of two quinolones were also evaluated on gastic mucus secretion by using the Alcian blue dye binding method and haematological parameters. Ciprofloxacin and pefloxacin showed a significant antiinflammatory activity and decreased the white blood cell count (WBC). However, there was no significant difference in the other haematological parameters. These two quinolones stimulated gastric mucus secretions, but these increases were not statistically significant. These findings suggest that ciprofloxacin and pefloxacin (at 50 mg/kg p.o. doses) possess antiinflammatory activities and are well-tolerated orally. Further detailed investigations are needed to clarify the mechanisms of their antiinflammatory activities.

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3′-Phosphoadenosine 5′-phosphosulfate (PAPS) and UDP-glucuronic acid (UDPGA) in cultured rat hepatocytes were directly assayed by isocratic reverse-phase HPLC. The sample preparation involves the extraction of nucleotide cofactors with cold alkaline solvent and clearing treatment by ultra-filtration. Nucleotides were separated on HPLC equipped with a RPAQUEOUS C-30 column adjusted to 20°C using 100 mM sodium potassium buffer (pH 5.5) as elution solvent and detected by UV absorption at 260 nm. Linear calibration curves were obtained over the ranges from 0.1 to 1.0 μM for PAPS and 1.0 to 10 μM for UDPGA in both distilled water and hepatic cell extracts. Peaks in the cell extracts were identified as PAPS and UDPGA by comparison of the retention times and co-elution with each of their corresponding authentic standards. Assignment of peak identity was additionally supported by the preferential decrease in PAPS and UDPGA in cultured hepatocytes which were incubated with quercetin or D-galactosamine. This HPLC method was found to be sensitive enough to accurately quantify the cellular contents of PAPS and UDPGA far below that normally found in cultured rat hepatocytes.

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A simple and rapid analytical method of oil-soluble coal tar dyes in cosmetics was established using reversed-phase TLC/scanning densitometry. Eleven kinds of oil-soluble coal tar dyes were able to be separated completely on reversed-phase TLC plates by the complementary use of 2 solvent systems. The solvent systems were A; n-hexane/2-butanone solution (5 : 1, v/v), solvent system B; acetonitrile/methanol solution (5 : 1, v/v). Then we measured visible absorption spectra of spots developed on the reversed-phase TLC plates by scanning densitometer to identify these coal tar dyes. The proposed method was successfully applied to the identification of oil-soluble coal tar dyes in commercial cosmetics.

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Sodium spirulan (Na-SP) is a sulfated polysaccharide with M_r ~220000 isolated from the blue-green alga Spirulina platensis. Na-SP influences the blood coagulation-fibrinolytic system by activation of heparin cofactor II in vitro, although it has been incompletely understood whether the polysaccharide can act on vascular endothelial cell functions. In the present study, we investigated the effects of Na-SP on the fibrinolytic activity of human coronary endothelial cells in a culture system. It was found that Na-SP enhances the activity of tissue-type and urokinase-type plasminogen activators in the conditioned medium of the cells through induction of urokinase-type plasminogen activator secretion and inhibition of plasminogen activator inhibitor type 1 (PAI-1) secretion. The inhibitory effect of Na-SP on PAI-1 secretion was maintained even when sodium ion was removed or replaced by calcium ion, while it disappeared with desulfation, indicating that metal ion is not required but the sulfate group is essential for the inhibition of endothelial PAI-1 secretion by Na-SP. The present study revealed that Na-SP not only shows a strong antithrombin effect through activation of heparin cofactor II but also exhibits a fibrinolytic property through differential effects on endothelial fibrinolytic protein secretion.

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The potential neurotoxic effects of diphenylarsinic acid (DPAA) was investigated using two in vitro assay systems, since DPAA could be formed from phenylarsenic compounds as chemical warfare agents in the environment and pose potential health risks to the population using ground water contaminated with DPAA as a source for drinking water. DPAA showed inhibitory effects on the neurite extension in human neuroblastoma NB-1 cells similar to that of methylmercury, although it required a several hundred times higher dose than that of methylmercury. In addition, DPAA affected the expression and localization of 440-kDa ankyrin_B, a neuron-specific marker protein, in rat cerebellar neurons in primary culture. These results suggest that DPAA may have weak neurotoxic actions similar to those of methylmercury, even though it requires a 1000 times higher dose than methylmercury.

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