Human milk is an important food source for infant because it contains a large number of nutritional substances, growth and immune factors. However, human milk may be contaminated with environmental pollutants when mothers are exposed to these pollutants. In particular, lipophilic organic pollutants are likely to accumulate in milk fat. Therefore, the determination of the organic pollutants levels in human milk is necessary to estimate the health risks of these pollutants to milk-fed infants. For this purpose, a lot of reports for the measurements of environmental pollutants in milk samples have been published. In this review, we summarized the concentrations of harmful organic environmental pollutants such as polychlorinated organic compounds (PCOCs), polybrominated compounds, perfluorinated compounds (PFCs), polycyclic aromatic hydrocarbons (PAHs) and endocrine disrupting phenols in human milk samples. Also, we described the noteworthy results of several evaluative studies such as time trend and regional difference of pollutant levels.

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We observed that maternal exposure to diesel exhaust (DE) and diesel exhaust particles (DEPs) damaged the reproductive and central nervous systems in mice and rats. These observations suggest that impairment of early development induced by maternal exposure to DE and DEP causes several disorders after growing up. To elucidate the effects of maternal exposure to environmental substances, we review here a hypothesis of fetal and early developmental origins of adult disease. Recent studies influenced by Dr. Barker's Thrifty Phenotype Hypothesis have led to advances in understanding how fetal and infant malnutrition can permanently and adversely alter the development of tissues and organs. Several epidemiological surveys in humans have uncovered links between maternal malnutrition and effects on the organs such as the kidney, pancreas, liver, muscles, adipocytes, and the hypothalamic-pituitary-adrenal (HPA) axis. These observations were examples of critical period programming. The idea has been applied to examining possible fetal and early origins of other diseases. Interestingly, many reports showed that similar phenomena were induced by perinatal exposure to airborne environmental pollutants. Studies have shown that maternal DE exposure disrupts reproductive development and damages the central nervous system. In addition, perinatal exposure to tobacco smoke has been linked to several respiratory disorders. These results show that early development is a critical determinant of adult physiology and much care should be taken to ensure the proper environment for fetal development. This idea is especially topical currently, where rapid industrialization in Asia has accelerated changes in environment and increased pollution.

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As screening methods to predict carcinogenicity, genotoxicity assays have a major issue in that many carcinogens are negative in such assays. Non-genotoxic mechanisms, which are at least initially independent of direct DNA damage, can play a causal role in carcinogenesis. Also, it is predicted that among these non-genotoxic carcinogens, many will be tumor promoters. There is therefore a need to develop tumor promoter assays, and in vitro assays have been developed to detect phenomena such as the inhibition of gap junctional intercellular communication, the promotion or inhibition of cell differentiation, the expression of Epstein-Barr virus early antigen (EBV-EA), and cell transformation assays. However, none of these assays has been adopted in the battery of official safety screening tests for chemicals. One reason is that some methods are not simple for routine screening. Given this lack, we established a novel short-term in vitro method to detect the tumor promoting potential of chemicals, the Bhas promotion assay. This is a cell transformation assay using Bhas 42 cells. It has many advantages compared with other focus formation (cell transformation) assays. Transferability and applicability of this assay was confirmed by an inter-laboratory collaborative study. Furthermore, our study has demonstrated that the Bhas promotion assay has practical utility value in monitoring the promoting potential of environmental contaminants.

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Trypanosoma cruzi (T. cruzi) is a parasitic protozoan transmitted to mammalian hosts by blood-sucking triatomine bugs. Infections by T. cruzi, known as Chagas' disease, pose a major public health problem in endemic countries in Central and South America. New chemotherapeutic agents are desired because of the lack of effective vaccines, undesirable side effects of anti-chagasic drugs in use such as nifurtimox and benznidazole, and the emergence of parasite resistance to these drugs. In the past two decades, novel advances and an improved understanding of the biology and biochemistry of T. cruzi have led to the identification of various targets for chemotherapy to treat Chagas' disease. In addition, many efforts have been undertaken to develop antichagasic agents, such as designed and synthesized compounds, natural products, and their derivatives, against a number of targets. Here, I mainly review recent studies on the antichagasic activities of natural products.

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Transgenic rodents are valuable models for investigation of genotoxicity of chemicals in vivo. We have developed gpt delta transgenic mice (C57BL/6J background) and rats (Sprague-Dawley, SD), which have the ability to identify both point mutations by the gpt assay [6-thioguanine (6-TG) selection] and certain types of deletions by the Spi^- (Spi, sensitive to P2 interference) assay. Recently, the gpt delta SD rat was backcrossed with the Fisher 344 (F344) rat to establish an gpt delta F344 rat. The average spontaneous gpt mutation frequencies (MFs) are about 4.5×10⁶ in both SD and F344 gpt delta rats as well as in gpt delta mice. The G:C to A:T transitions at 5'-CpG-3'sites and G:C to T:A transversions are the predominant spontaneous gpt mutations in rats and mice. However, there is one false mutation (e.g. A:T to T:A at position 299) in the rats. The base substitution may have arisen when the lambda EG10 transgene was introduced into the genome of the SD rat during transgenesis. In the Spi^- assay, 1-bp deletions in repetitive sequences are predominantly observed in both mice and rats. Possible mechanisms underlying the spontaneous mutations in gpt delta rodents are discussed.

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In order to measure the level of toluene in the blood of fetuses of pregnant rats exposed to toluene, application of headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was studied. Pregnant rats from gestational day 15 (GD15) to GD19 were subjected to inhalational exposure to toluene for 90 min per day. They were obtained by Cesarean section on the fifth day of exposure (on GD19), and the level of toluene in the blood of mother rats and in fetuses were quantified using 5 μl blood. The levels of toluene in the blood of the fetuses in the groups exposed to toluene at 9 ppm and 90 ppm were 0.07±0.03 μg/ml (n=17) and 2.0±0.51 μg/ml (n=16) respectively, which were significantly higher than the blood level of the fetuses in the control group (0.02±0.02 μg/ml, n=12). Meanwhile, the blood concentrations of mother rats were 0.06±0.06 μg/ml (n=3, control group), 0.14±0.03 μg/ml (n=3, 9 ppm exposure group), and 3.5±1.5 μg/ml (n=4, 90 ppm exposure group), respectively, which were higher than those of the fetuses per unit blood volume.

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Chicken soup has long been considered anecdotally healthful in Western and Southeast Asian countries. In this study, we examined the effects of a 2-week intake of chicken soup on mood states and of a single and 2-week intake on peripheral blood flow. Thirty healthy volunteers (7 men and 23 women aged 20.5±1.4 years) participated in a randomized, double-blind, placebo-controlled crossover study with 2 weeks treatment and washout periods. They were randomly assigned to two groups, and daily received either chicken soup or placebo soup. Mood states by the Profile of Mood States (POMS) questionnaire and peripheral blood flow by a laser-Doppler blood flow imaging system were assessed before and after each treatment period. On the first day of the treatment periods, the effect of single intake on peripheral blood flow was investigated. The 2-week intake of chicken soup significantly reduced the tension-anxiety (T-A) score compared to the placebo soup (p<0.05). The single intake of chicken soup significantly increased peripheral blood flow as compared with the base value (20 min after intake p<0.01; 25, 30 and 45 min after intake p<0.001) and that of the placebo soup (25, 30 and 60 min after intake p<0.05; 45 min after intake p<0.01). The 2-week intake of chicken soup also significantly increased peripheral blood flow over that of the placebo soup (p<0.001). Chicken soup was considered to have improved mood states such as tension and anxiety and increased peripheral blood flow.

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We attempted to perform multilevel analyses to explore whether a year-to-year weight variation causes any corresponding effects on blood pressure (BP) among middle-aged Japanese workers. Subjects were 4547 healthy male workers 40-59 in age from whom serial data of systolic and diastolic BP were collected during health checkups conducted in the years 1997-2000. The effects on BP of a time-varying body mass index (BMI) that was rescaled to center around the individual-specific mean of 4 examinations on BP were investigated by statistical analysis with multilevel modeling to adjust for the wide variability among individuals. A significant relationship between the time-varying BMI and both systolic and diastolic BP was confirmed. None of the interaction terms for BMI×potential effect modifiers (smoking history, drinking status, leisure-time physical activity, and preference for salty taste) included in the subanalysis model showed any significant modifying effect. Our result indicated that the year-to-year weight variation, though usually in a much narrower range than the between-individual variation, has a strong impact on the corresponding BP. This result supports the public health significance of interventions in short-term weight control to prevent the development of hypertension among an otherwise healthy workplace population.

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Cytosolic zinc-binding protein, metallothionein (MT), is normally saturated with Zn. It is thought that Zn-saturated MT (Zn-MT) acts as a major intracellular Zn pool. Metal-response element-binding transcription factor-1 (MTF-1) plays an important role in Zn-mediated MT transcription. Here, we showed that degradation of Zn-MT activates MTF-1. We measured activated MTF-1 using an electrophoretic mobility shift assay. Interleukin-6 induced MT expression and increased MTF-1 activity. MTF-1 activation was not observed in MT-overexpressing cells. MT-dependent MTF-1 activation was observed only after treating MT-overexpressing cells with cycloheximide (CHX), a protein synthesis inhibitor. CHX-treatment increased the degradation/synthesis ratio of protein. An increase in the degradation/synthesis ratio for the MT protein is expected to increase the level of labile Zn and activate MTF-1. Recombinant MTF-1 was activated by H₂O₂ only in the presence of Zn-MT. Oxidative stress activated MTF-1 DNA-binding activity in primary cultured hepatocytes but not in MT-deficient hepatocytes. These findings suggest that degradation of Zn-MT activates MTF-1, and that MT plays an important role in zinc-mediated signal transduction.

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We developed a sampler for collecting atmospheric polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) by attaching a glass column packed with XAD-4 resin to the gas sampling port of a high-volume air sampler equipped with a filter. When the upper and bottom layers of the column were packed with 64 g and 32 g of XAD-4 resin, respectively, all PAHs and NPAHs in the gas phase were quantitatively collected in the XAD-4 resin column without any break through, while PAHs and NPAHs in the particle phase were collected on the filter. We collected air samples at suburban and downtown Kanazawa by using the proposed sampler. It was found that about 95% of 2-3 ring PAHs and more than 99% of 2-ring NPAHs existed chiefly in the gas phase, that 4-ring PAHs such as fluoranthene (FR) and pyrene (Pyr) and 3-ring NPAHs were in both the gas and particle phases and that the other PAHs and NPAHs having 4-rings or more except for FR and Pyr were almost completely in the particle phase. Our data also indicated that the adsorption of NPAHs to the particle phase in the atmosphere is controlled by the same mechanism as that of PAHs.

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Ferrous ferric chloride (FFC) is a special form of aqueous iron that is a complex of ferrous chloride and ferric chloride and participates in oxidation and reduction reactions. My previous study showed that FFC stimulated the proliferation and differentiation of cultured epidermal melanoblasts or melanocytes derived from newborn mice. However, it is not known whether FFC stimulates the proliferation and differentiation of melanocytes in cooperation with natural factors, such as vinegars, vitamins, and herbal medicines. The drink Pairogen^® (Akatsuka Co., Tsu, Japan) consists of FFC, vinegars, and vitamins, while Pairogen Gold^® (Akatsuka Co.) contains herbal medicines in addition to FFC, vinegar, and vitamins. To clarify whether these natural factors supplemented to Pairogen or Pairogen Gold elicit stimulative effects on skin function, Pairogen and Pairogen Gold were added to the culture medium and tested for their proliferation- and differentiation-stimulating activity on melanocytes. Although Pairogen Gold failed to increase melanocyte proliferation beyond that elicited by FFC alone, it markedly increased melanocyte differentiation. In contrast, Pairogen possessed no such effect. The extracts of Chinese wolfberry (Lycium chinense) and Siberian ginseng (Eleutherococcus senticosus) that are included in Pairogen Gold stimulated melanocyte differentiation additionally with FFC. Therefore, these results suggest that FFC is involved in regulating the differentiation of melanocytes additionally with extracts of Chinese wolfberry and Siberian ginseng.

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Nanomaterials are being used increasingly for commercial purposes, yet little is known about the potential health hazards such materials may pose to consumers and workers. Here we show that nano-sized titanium dioxide (TiO₂), which is used widely as a photo-catalyst and in consumer products, administered subcutaneously to pregnant mice is transferred to the offspring and affects the genital and cranial nerve systems of the male offspring. Nanoparticles identified as TiO₂ by energy-dispersive X-ray spectroscopy were found in testis and brain of exposed 6-week-old male mice. In the offspring of TiO₂-injected mice, various functional and pathologic disorders, such as reduced daily sperm production and numerous caspase-3 (a biomarker of apoptosis) positive cells in the olfactory bulb of the brain, were observed. Our findings suggest the need for great caution to handle the nanomaterials for workers and consumers.

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A particle-counting aggregometer employing laser-light scattering was used in systematic manners to screen and to detect inhibitor(s) of platelet aggregation from the extract of Malbranchea filamentosa (M. filamentosa) IFM41300. The inhibitor was determined as adenosine on the basis of the ¹³C- and ¹H-NMR spectral data. Although adenosine was previously reported as an inhibitor of platelet aggregation, we isolated the compound from the fungus for the first time. Because the method was able to provide us with information on the developmental formation of platelet aggregates in different sizes with incubation time, we further elaborated the inhibitory behaviors of adenosine, as an example, at varied concentrations in different solvents such as dimethyl sulfoxide (DMSO) and saline. We found that DMSO could facilitate to dissolve less water soluble materials from herbs and fungi by using the present assay method.

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Chemical models for cytochrome P450, consisting of an iron porphyrin complex and an oxidant, have been used as substitutes for the S-9 mix for detecting mutagenicity of promutagens in the Ames assay. In this study, we developed optimized procedures for the Ames mutation assay using a water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F₅P) or a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron(III) (4-MPy) plus tert-butyl hydroperoxide (t-BuOOH) as a chemical model to determine 2-aminofluorene (AF) mutagenicity. The model system including the water-insoluble F₅P plus t-BuOOH demonstrated higher AF mutagenicity when the tester strain was added following the incubation period of the reaction mixture. In contrast, in the system including the water-soluble 4-MPy plus t-BuOOH, the activity of AF mutagenicity was highest when the tester strain was added to the reaction mixture prior to incubation. It is thus possible to detect short-lived mutagenic metabolites by the latter procedure. AF mutagenicity was compared among diverse water-soluble iron porphyrins plus t-BuOOH. The results showed that a cationic 4-MPy/t-BuOOH had the highest capacity for mutagenic activation of AF among the chemical models tested.

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Current risk assessment methods for environmental chemicals are based on adult physiology. However, recent reports have shown an increased incidence of neurodevelopmental disorders which may result from exposure to chemical in utero and during the early postnatal period. We previously showed that exposure of neonates to the environmental chemical p-nitrotoluene caused hyperactivity, accompanied by changes in the expression of the mesencephalic dopamine transporter gene. In this study, we have examined the effects of p-nitrotoluene on cultured neural stem cells isolated from the rat mesencephalon. At embryonic day 15 (E15), these cells stained positive with antibodies against nestin, microtubule-associated proteins (MAPs), and glial fibrillary acidic proteins (GFAPs). The treatment of cultured neurospheres with p-nitrotoluene (1 μM; 72 hr) facilitated differentiation with two distinct morphologies outside the sphere, being neural and glial lineages. Neurospheres could therefore be used as a very simple primary assay for screening environmental chemicals for disruption of developmental programming.

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17β-Estradiol (E2) and bisphenol A (BPA) damage DNA in estrogen receptor (ER)-positive human mammary tumor MCF-7 cells. The effect of E2 and BPA is associated with increased expression of Bloom helicase (BLM) and formation of nuclear foci consisting of BLM and γH2AX at double stranded break regions. BLM is one of five human RecQ helicases that participate in the maintenance of genomic stability. This study investigated if the expressions of RecQ helicases Werner syndrome helicase (WRN), Rothmund-Thomson syndrome helicase (RTS), RecQ5 helicase (RecQ5), RecQ protein-like 1 helicase (RecQL1, also known as RecQ1) and BLM helicase are affected in MCF-7 cells by treatment with estrogenic agents. The expression of RTS mRNA significantly increased although the expression in RTS protein only slightly increased. The expressions of WRN, RecQ5 and RecQL1 were almost unaffected both at mRNA and protein levels. These data suggest that the expression of human RecQ helicases is differentially regulated depending on the tissues and cells. We showed that some helicase expressions are regulated depending on the DNA damage.

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Based on Intergovernmental Panel on Climate Change (IPCC) assessment report at 2007, it is likely that there has been a substantial anthropogenic contribution to global warming. Carbon dioxide (CO₂) is a major anthropogenic greenhouse gas and its increase is thought to give rise to the recent global warming. Although studies suggested the impact of population growth on carbon dioxide increase, much attention has not been paid. In this study population was plotted as compared to the atmospheric CO₂ concentration. A quite linear relationship was observed between population and CO₂ concentration at both before and after 1970, after which the global temperature rapidly increased. In addition, direct and indirect human-derived CO₂ emission appeared to contribute much to the total amount of CO₂ emission in developing countries and as the economy grow fossil-fuel-derived CO₂ emission increased more as compared to human-derived emission. These findings indicate that population growth especially in developing countries is a critical factor for manipulation of global CO₂ increase.

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The quantity of citrate was determined using apparatus comprised of a reactor with immobilized citrate lyase and malate dehydrogenase in the flow line. The decrease of β-nicotinamide adenine dinucleotide, reduced form by enzymatic reactions was spectrophotometrically detected as a negative peak. The maximum peak area was obtained at pH 7.6 when the pH of the carrier consisting of phosphate buffer ranged from 6.6 to 8.0. Various buffer types were also examined as carrier media at pH 7.6 and phosphate buffer showed the maximum peak area. When the carrier composed of phosphate buffer (0.1M, pH 7.6) was used, the calibration curve for citrate was linear in the range 0.5-100 μM (r=1.000). The detection limit (S/N=3) was 0.2 μM. The relative standard deviation of the peak area at 10 μM was 2.2% (n=7). This method was applied to analyze citrate in beverages, and citrate content determined by this method agreed with that determined by a commercially available test kit.

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Relaxin-3 is a recently discovered member of the insulin superfamily and is a ligand for three orphan G-protein-coupled receptors: GPCR135, GPCR142 and LGR7 (leucine-rich repeat-containing G-protein-coupled receptor 7). GPCR135 mRNA is expressed in the pancreas, however, it is not known, whether the peptide affects pancreatic islet function. Reverse transcriptase (RT)-PCR and radioreceptor assay have shown that the relaxin-3 receptor (GPCR135) is expressed in pancreatic islets and rat insulinoma, but LGR7 is not expressed. Moreover, relaxin-3 has been revealed to inhibit the secretion of insulin from pancreatic islets. However, we can not detect relaxin-3 in small intestine and pancreas. These results suggest a novel role of relaxin-3 in the regulation of insulin release.

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Inhibition of histone deacetylase (HDAC) modulates the expression of many genes and induces cell cycle arrest, apoptosis, and differentiation in several cancer cell lines. Melanoma is a malignant phase of cutaneous melanocytes originally derived from the neural crest and is highly metastatic. A therapeutic agent capable of inducing differentiation of metastatic melanoma cells into a nonmalignant stage would be useful to prevent metastasis. We examined whether HDAC inhibitors can induce differentiation of the murine melanoma cell line B16-BL6 into neural-type cells in vitro. A morphologic change accompanied by extended dendrites was induced in melanoma cells after treatment with the HDAC inhibitors butyrate and trichostatin A (TSA). The altered morphology was similar to that of neural cells. Many of the extended dendrites fused with other dendrites of neighboring cells and had synapse-like knobs on their dendrites. These dendrites showed positive labeling with anti-α/β-tubulin and anti-L1 cell adhesion molecule (L1CAM) antibodies but were seldom stained with phalloidin, suggesting that the neural cell-related proteins were major components of the extended dendrites but that actin stress fibers were not. Furthermore, the mature neuron-specific cytoskeletal protein, microtubule-associated protein 2 (MAP2), was detected with the specific antibody in the extended dendrites of cells treated with butyrate and TSA. Butyrate treatment increased the levels of MAP2 and neural cell adhesion molecule (NCAM) mRNA expression in the cells. However, the treatment did not alter the expression of neurofilament light chain (NF-L) mRNA. The observations suggest that the differentiated cells were neural-type cells but not complete neural cells.

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The effectiveness of spherosomes for reducing pesticide accumulation in cucumbers and for the control of Aphis gossypii Glover was studied. Spherosomes could efficiently reduce the uptake of imidacloprid in cucumbers. In the case of imidacloprid with spherosomes, the number of Aphis gossypii Glover, surprisingly, became zero after 3 and 7 days, and showed almost the same effect as imidacloprid without spherosomes, although the concentration of imidacloprid in cucumbers decreased by about 1/5 in comparision with that without spherosomes. It seems more likely that spherosomes might have a role as adsorbent materials for removal of imidacloprid in soil. The application of spherosomes to farmland is a useful and effective method to keep the concentration of pesticides in crops at a low level, resulting in sustainable agriculture.

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