Lead and cadmium are toxic heavy metals that have been shown to be possible risk factors of atherosclerosis in epidemiological and experimental studies. Since excess extracellular matrix, including proteoglycans, accumulates changing the composition and the structure in the atherosclerotic vascular wall, the effects of lead and cadmium on the proteoglycan synthesis in vascular cells have been studied using a cell culture system. The following results were obtained: Lead does not destroy endothelial cell layers but markedly inhibits the repair of the injured cell layers, which results from a lower response to endogenous basic fibroblast growth factor caused by inhibition of the synthesis of perlecan, a large heparan sulfate proteoglycan, in vascular endothelial cells. Lead selectively inhibits the synthesis of versican, a large chondroitin sulfate proteoglycan, in vascular smooth muscle cells only at a high cell density. Cadmium induces the synthesis of biglycan and decorin, small dermatan sulfate proteoglycans, in vascular smooth muscle cells only at a low cell density, while inhibiting the synthesis of other proteoglycan molecules. It is therefore suggested that lead and cadmium may influence the developmental process of atherosclerosis through disrupting the regulation of proteoglycan synthesis in vascular cells.

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For the risk management of chemical substances in foods, evaluating the exposure to chemical substances is essential. The Ministry of Health, Labour and Welfare (MHLW) continuously performs total diet studies to estimate the average Japanese dietary intake of various chemical substances, such as pesticides, heavy metals, dioxins, food additives, etc. In many of the studies, the National Institute of Health Sciences, especially the Division of Foods plays a central role. In this mini-review, the results of several recent total diet studies are described. The Total Diet Study of Food Contaminants which began in 1977 has found that the intake of banned pesticides is very low compared with the acceptable daily intake (ADI). However, the dietary intake of Cd is high, and was 52% of the Provisional Tolerable Weekly Intake in 2002. The average daily intake of dioxins [polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar-polychlorinated biphenyls (PCBs)] was 1.49 pgTEQ/kg in 2002, which is lower than the tolerable daily intake (TDI) value of 4 pgTEQ/kg/day. Total diet study samples from the Kansai area demonstrated a reduction in dioxin intake between 1977 and 2002. The daily dietary intake of acrylamide was estimated to be several scores of micrograms per person. Although the daily intake of most food additives was much lower than ADI, the intake of nitrate exceeded ADI when estimated by the market basket method. The contribution of food additives was low and most of the intake was attributed to nitrate in vegetables. These data form the basis for risk management performed by the MHLW.

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Several reports have suggested that semen quality in normal men is declining. Given the lack of consensus about the effects of diesel exhaust (DE) on the male reproductive system, we conducted various experiments. We examined the effect of the exposure of mature male mice to DE for 6 months on the male reproductive system. Daily sperm production per gram from the testes dose-dependently decreased with exposure to DE. Next, we investigated the effect of exposure of pregnant mice to DE on male gonad development at the level of mRNA expression. Expression of mRNAs for steroidogenic factor (Ad4BP/SF-1) and Müllerian inhibiting substance (MIS) decreased significantly in male fetuses when maternal mice were exposed to DE for 8 hr per day between days 2 and 13 post coitum. In addition, DE exposure during the fetal period may have some influence on the male reproductive function in newborn mice. In utero DE exposure caused testosterone levels to increase in newborn male mice at age 4 weeks. These findings indicate that exposure to DE may influence the male reproductive system. Further studies are necessary to elucidate the mechanism of the effects of DE on the male reproductive system and to define which classes of compounds are responsible for the changes in the male reproductive system.

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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR is a nucleo-cytoplasmic shuttling protein mediated by nuclear localization signals (NLS) and nuclear export signals (NES). Protein kinase C-mediated phosphorylation of NLS inhibits the ligand-dependent nuclear import of AhR, suggesting a two-step mechanism of nuclear import. Cell density regulates AhR intracellular localization, due to modulation of nuclear export activity. The p38 mitogen-activated protein kinase (MAPK)-mediated phosphorylation of NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization.

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Long-term toxicity growth tests were conducted with DDT using zebrafish (Danio rerio). The fish were exposed to concentrations of 0.05 μg/l, 0.5 μg/l, 5 μg/l and 50 μg/l DDT under flow-through conditions. The effect of the compound on the ecologically important parameters hatching rate, duration of the developmental stages and growth were the focus of interest. When exposed to 50 μg/l DDT, the hatching rate was 2 to 3 times lower than in embryos exposed to 0.05 μg/l DDT. This study demonstrates that the rearing zebrafish in the pesticide causes a reduction in body length and the durations of the life cycle stages (LCS) of the offspring are extended. These effects are increased when the fish are exposed to higher DDT concentrations.

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

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An investigation was conducted to determine both the distribution and the concentration of 24 types of disinfection by-products (DBPs) formed by the chlorination of tap water during the water treatment process. The 24 DBPs consisted of 9 haloacetic acids (HAAs), 9 haloacetonitriles (HACNs), 4 trihalomethanes (THMs), chloral hydrate (CH) and formaldehyde (FA). The samples were collected from 8 different water sources in the Hyogo Prefecture. As a result of the field study, which was conducted 4 times in one year, 23 of the DBPs mentioned above [all except for tribromoacetonitrile (TBACN)] were detected in tap water. When the 24 DBPs are classified into their 5 main categories, it has previously been thought that THMs would form the highest concentration group. However, according to the results obtained in this study, the average values of the HAAs showed the highest at the various sampling points, followed by the THMs, HACNs, chloral hydrate and formaldehyde. The composition ratios of the bromine/chlorine-containing DBPs in tap water from different water sources were compared. The ratio of chlorine-containing DBPs in tap water derived from surface water was higher than that in tap water derived from ground water. On the other hand, the ratio of the bromine-containing DBPs in tap water extracted from ground water was higher than that of tap water extracted from surface water. This tendency was observed equally for HAAs, HACNs and THMs. This phenomena was also supported by numerical results obtained using the bromine incorporation factor [n(Br)]. It was newly revealed that n(Br) is applicable to HAAs and HACNs in addition to THMs.

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The immunotoxicological effect of 17β-estradiol and other estrogenic compounds was determined with respect to lymphocyte apoptosis. High concentrations (10^-6-10^-5 M) of 17β-estradiol inhibited lymphocyte proliferation. DNA fragmentation was observed by agarose gel electrophoresis when lymphocytes were exposed to 17β-estradiol, confirming the occurrence of apoptosis. After exposure to 17β-estradiol, the lymphocytes underwent apoptosis within 12 hr of culture through three sequential alterations of membrane structure, as judged by the results of staining with three kinds of dyes, namely annexin V, which binds on the surface of the cell membrane, and propidium iodide and trypan blue, which cross the cell membrane. Diethylstilbestrol killed more cells whereas bisphenol A, a weak estrogen, induced apoptosis in fewer cells. These results suggest that 17β-estradiol and other estrogenic compounds induce lymphocyte apoptosis.

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To isolate the genes regulated by peroxisome proliferator-activated receptor γ (PPARγ), we first developed a stable transformant expressing PPARγ. Using cytosol and nuclear fractions prepared from this cell line, we performed two-dimensional electrophoresis. A comparison of the electrophoretic patterns with those from control cells revealed many spots to be up-regulated or down-regulated proteins. Some spots were subjected to mass spectrometric analyses, and triosephosphate isomerase and galectin-3 were identified as proteins down-regulated by PPARγ. Moreover, Northern blot analyses revealed that the expression level of both genes decreased in PPARγ-expressing cells. These results strongly suggest that triosephosphate isomerase and galectin-3 are target genes whose expression is mediated by PPARγ.

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We carried out a dimethylamphetamine (DMA) smoking experiment that is closer to some real cases. In this study, we heated DMA hydrochloride (DMA-HCl) in the range of about 250°C to 350°C, in which demethylation reaction occurred mainly, with a smoke collection apparatus and gas lighter, and trapped the generating vapor with an adsorption cartridge. The eluate desorbed from the cartridge and residual materials inside the smoke collection apparatus were analyzed by gas chromatography-mass spectrometry and liquid chromatography-electrospray ionization-mass spectrometry. Methamphetamine (MA) and amphetamine (AM) were produced via demethylation from the dimethylamino group of DMA. Allylbenzene (AB), benzaldehyde (BA), cis-β-methylstyrene (cMS), benzyl chloride (BC) and trans-β-methylstyrene (tMS) in addition to MA and AM, were also formed as pyrolysis products. The molar percentages of a pyrolyzate MA and AM to the starting DMA were 21.4 and 1.8%, respectively, and the total molar percentage of AB, BA, cMS, BC and tMS was 2.9%. Taking into account the central nervous system stimulation by DMA and MA in humans, the total stimulant effects of the drugs that are ingested in a body can be calculatedly increased more than fourfold by smoking DMA. The present data show that a DMA smoking abuser may be able to get a more powerful stimulant effect by smoking than by injecting.

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We evaluated 8 clinically-used non-steroidal anti-inflammatory drugs (NSAIDs): Aspirin, Etodolac, Diclofenac, Ibuprofen, Indomethacin, Mefenamic acid, Nabumetone, and Piroxicam, for cytotoxicity in the human bladder cancer cell line UM-UC-3 in vitro. Basing our dosages on the maximum concentration of each NSAID used clinically, four concentrations of each drug were prepared, and the viability of UM-UC-3 cells given each treatment was monitored for up to 5 days. At the highest concentration, Aspirin and Diclofenac caused no decrease in UM-UC-3 viability during the 5-day incubation. Etodolac, Ibuprofen, Indomethacin, and Piroxicam decreased the viability of UM-UC-3 cells about 20% on the fifth day. Treatment with 30 μg/ml of Mefenamic acid and 16 μg/ml of Nabumetone caused about a 40% loss of cell viability on the last day of the experiment. Thus, the most effective cytotoxicity was observed with Mefenamic acid and Nabumetone. We then examined the nature of the cell death induced by these two drugs, by biochemical and morphological analyses. A DNA fragmentation assay showed DNA-ladder formation with both Mefenamic acid and Nabumetone treatment. In addition, nuclear blebbing was observed by fluorescence microscopy in both Mefenamic acid- and Nabumetone-treated cells. Since DNA ladder formation and nuclear blebbing are hallmarks of typical apoptotic cell death, we concluded that at least two NSAIDs, Mefenamic acid and Nabumetone, could cause apoptotic cell death in the human bladder cell line, UM-UC-3.

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Iron acquisition is a fundamental requirement for many aspects of life, but excess iron may result in the formation of free radicals that damage cellular constituents. Therefore, the amount of iron within the cell is carefully regulated by the iron metabolism (IRE/IRP system) in order to assure an adequate level. We previously reported that long-term excessive L-methionine consumption increases iron and lipid peroxide levels in rat liver. To determine whether such excess iron accumulation depends on the elevation of transferrin receptor via oxidative stress, we investigated the possible effects of long-term excessive L-methionine intake on the iron metabolism and the mitochondrial function in rat liver. Wistar male rats were fed either an L-methionine-supplemented (16.0 g/kg) diet or a control diet for 3 and 6 mo. The expression of transferrin receptor is significantly elevated by excess L-methionine intake, indicating that an accumulation of iron may be accompanied by such an elevation in the liver. Long-term excessive L-methionine consumption significantly decreases the H₂O₂ production of rat liver mitochondria without inducing changes in mitochondrial oxygen consumption. No significant differences in either glutathione peroxidase activity or superoxide dismutase activity were shown between the two groups. In contrast, the glutathione concentration significantly increased in L-methionine-treated rats compared to controls. These results indicate that long-term consumption of excess L-methionine by rats may affect mitochondrial function, resulting in a reduction in H₂O₂ generation. Moreover, an accumulation of iron by excess L-methionine intake may be responsible for a mechanism other than the IRE/IRP system via mitochondrial oxidative stress.

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The optimum conditions for the enzymatic hydrolysis of α-hydroxytriazolam (α-OHTRZ)-glucuronide, one of the major metabolites of triazolam in human urine, were determined. β-Glucuronidases from Escherichia coli (E. coli), bovine liver, Helix pomatia (H. pomatia) and Patella vulgata (P. vulgata) were used, and the parameters studied were amounts of enzyme used, temperature and pH range. α-OHTRZ was extracted with hexane/dichloromethane (1 : 1, v/v) and quantified using a high-performance liquid chromatography with a UV detector set at 230 nm. A preliminary study showed that β-glucuronidase from H. pomatia gave a poor recovery compared with the other three enzymes. The optimal conditions (amounts of enzymes, temperature and pH range) for 1 ml of urine were as follows; β-glucuronidase from E. coli (100 U, 37°C, pH 5.5-7.8), bovine liver (100 U, 45°C, pH 5.0-5.5), and P. vulgata (300 U, 60°C, pH 3.8-4.5). Among these enzymes, β-glucuronidase from E. coli was the most effective for the hydrolysis of α-OHTRZ-glucuronide in terms of efficiencies and the wide pH range tolerated. Incubation for 90 min with β-glucuronidase from E. coli was sufficient for hydrolysis of α-OHTRZ-glucuronide at clinical dose. α-OHTRZ-glucuronide in human urine can be hydrolyzed rapidly and effectively using this method.

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Airborne particulate samples were collected and fractionated into five sizes (diameter of < 1.1, 1.1-2.0, 2.0-3.3, 3.3-7.0, > 7.0 μm) with a high volume cascade impactor. The size distribution of airborne particulates was bimodal, having two peaks, coarse (> 7.0 μm) and fine (< 1.1 μm), which accounted for approximately 29% and 42%, respectively. Inhalable particulates accounted for approximately 50% of the total. The mass percentages of these extracts were also similar to airborne particulate concentrations. Eight authentic polycyclic aromatic hydrocarbons (PAHs; fluoranthene, pyrene, benz[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k] fluoranthene, benzo[a]pyrene, benzo[ghi]pelyrene) were detected in airborne particulates. The concentration of PAHs was dependent on size, with the following order: airborne particulates larger than 7.0 < 3.3-7.0 < 2.0-3.3 < 1.1-2.0 < smaller than 1.1 μm. Almost all of the PAHs detected existed in fine airborne particulates smaller than 1.1 μm. Ionic species in size-classified airborne particulate were also investigated. NH₄⁺, SO₄^2- and K⁺ existed in the fine particulates, whereas NO₃^-, Cl^- and Na⁺ existed in the coarse particulates. It is likely that SO₄^2- and NO₃^- were generated from the combustion of fossil fuel and air, and Na⁺ and Cl^- from sea salt. Since NO₃^- was generated from atmospheric NO₂, these concentrations were related to each other. The sampling site is located near a highway, so the results suggested that the main source of most fine particulates was car exhaust, especially diesel exhaust.

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Cadmium (Cd) is a well-known modulator of intracellular signal transduction including mitogen-activated protein kinases and protein kinase C. In this study, we investigated activation of a kinase using myelin basic protein (MBP) as a substrate during Cd-induced apoptosis in human leukemia HL-60 cells. To detect a kinase during Cd-induced apoptosis in HL-60 cells, we performed an in-gel kinase assay using MBP as a substrate and found that Cd induced the activation of a kinase with an apparent molecular mass of 36 kDa. The time-course of appearance of DNA ladders induced by Cd was consistent with that of activation of this kinase. The kinetics of activation of p36 MBP kinase was different from that of p38 mitogen-activated protein kinase (p38MAPK). Activation of p36 MBP kinase was also observed with kinetics distinct from that of activation of p38MAPK during mercuric chloride-induced apoptosis. This is the first report on activation of p36 MBP kinase during Cd- or Hg-induced apoptosis.

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An enzyme-linked immunosorbent assay (ELISA) was validated for the measurement of vitellogenin (VTG) in the cyprinid model species, medaka (Oryzias latipes). Both polyclonal antibody- and monoclonal antibody-based kits showed very high correlation between VTG values in unknown samples when we used standardized VTG protein. This was confirmed by 5 laboratories which participated in the ring test. The use of one gold standard VTG is essential to measure VTG values using ELISA kits in future OECD ring tests to screen for estrogenic chemicals in the 3 fish species medaka, zebrafish and fathead minnow.

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Loss of estrogen caused by ovariectomy (OVX) stimulates bone resorption and bone marrow B-lymphopoiesis, resulting in a marked bone loss and an accumulation of B cells in mouse bone marrow. In OVX mice, the expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA was elevated in trabecular bone and bone marrow compared with sham mice. To examine the roles of B-lymphocytes in bone resorption, B cells were purified from bone marrow, fixed, and co-cultured with mouse osteoblasts. Most of the fixed B cells adhered to cell surface of osteoblasts. The expression of RANKL mRNA in osteoblasts was markedly elevated by the contact with the fixed B cells, and the induction rate of RANKL was correlated with the number of B cells added. Treatment with inhibitors of ERK 1/2 and p38 MAP kinases suppressed the B cell-induced RANKL expression in osteoblasts, suggesting the involvement of these kinases in the signals via the cell-to-cell contact. These findings emphasize the roles of B-lymphocytes in RANKL-induced osteoclastogenesis and in pathogenesis of bone loss due to estrogen deficiency.

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Bisphenol A (BPA), a monomer used for the production of polycarbonate, is known to have estrogen activity. In this study, pregnant mice were fed chow diet containing 1% BPA, and we examined the influence of dietary BPA on delivery and growth of newborn pups in mice. When pregnant mice were fed BPA diet, the mice normally delivered pups on day 21 of gestation. The number of offspring pups in maternal mice fed BPA diet was same to those fed normal diet. Therefore, the growth of fetuses and the process of delivery were not influenced by dietary BPA. However, the growth of newborn pups was markedly suppressed when maternal mice were fed BPA diet. The growth of neonatal mice depends on breast milk, and stomach was filled with milk in pups. In newborn pups from maternal mice supplemented with BPA diet, the weight of stomach was lower than that from maternal mice with normal diet. Since the serum level of prolactin was limited in maternal mice supplemented with BPA diet, the suppressed growth of newborn pups by dietary BPA may be due to the insufficient supply of breast milk. These results suggest that the influence of BPA on the growth of newborn pups is related to hormonal condition in maternal mice.

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The color of urine from rats fed 1,4-dichloro-2-nitrobenzene (DCNB) is a clear yellow than that of urine from control rats. In order to obtain information on the relationship between the yellow color of the urine and its constituents, analyses of the components in the urine from rats fed DCNB were performed by UV, nuclear overhauser effect (NOE) method of ¹H-NMR and liquid chromatography (LC)-MS/MS. The urine from the rats fed DCNB had absorbance at a wavelength of 383 nm. Characteristic fragment ions of the N-acetylcysteine conjugates, at m/z 187, were clearly observed in the fragmentation of the precursor ions at m/z 317 ([M-H]^-) by LC-MS/MS. And position of the structure was found to be N-acetyl-S-(4-chloro-3-nitrophenyl)-L-cysteine by NOE method. Consequently, in view of the conformity of the major constituent (N-acetylcysteine conjugates), when comparing yellow urine and the metabolite, it is thought that the metabolite formation is attributable to the longer wavelength absorbance of the N-acetyl-S-(4-chloro-3-nitrophenyl)-L-cysteine, as compared to the control urine.

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