Teruyuki Nakao

Comparison of polybrominated diphenyl ethers in fish, vegetables, and meats and levels in human milk of nursing women in Japan

At present, little is known about the occurrence of polybrominated diphenyl ethers (PBDEs) in fish and food products sold in Japan. To investigate whether human exposure to PBDEs through the diet is significant, the concentrations of PBDEs were determined in fish and in meat and vegetables sold in two food markets in the city of Hirakata, Osaka prefecture. The concentrations of PBDEs in the breast milk of 12 primiparae nursing women at one month after delivery also were determined to ascertain the relationship between the levels found in the diet and levels in humans. sigma PBDE concentrations ranged between 21 and 1650 pg/g fresh weight in the edible tissues of five species of fish and one shellfish species. The highest concentrations were measured in yellow-fin tuna, followed by short-necked clam, salmon, yellowtail, mackerel and young yellowtail. Interestingly, sigma PBDE concentrations were not statistically significantly different in two cultured mackerel samples from Japan and mackerel collected from northern European waters. sigma PBDE concentrations in beef, pork and chicken meat (ranging between 6.25 and 63.6 pg/g fresh weight) and in three different vegetables (ranging between 38.4 and 134 pg/g fresh weight) were significantly lower than the concentrations in fish. In human milk, sigma PBDE concentrations ranged between 668 and 2840 pg/g lipid basis, which is comparable to the levels in populations of nursing women reported in Sweden and elsewhere. There was a strong positive relationship between PBDE concentrations in human milk and dietary intake of fish and shellfish, which was established in the women from responses to a questionaire on food consumption habits. The results of this study of food products commonly consumed in Japan and the levels found in nursing women raise a concern about low level PBDE contamination of fish and other foods intended for human consumption.

Assessment of human exposure to PCDDs, PCDFs and Co-PCBs using hair as a human pollution indicator sample I: Development of analytical method for human hair and evaluation for exposure assessment.

Dioxins including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and co-planar polychlorinated biphenyls (Co-PCBs) are highly toxic. Even at environmental pollution levels, they cause hormonal damage in women, and they have been shown to induce immunosuppression and genital function damage in humans. In this study, a new method using isotope dilution was established to detect PCDDs, PCDFs and Co-PCBs in human hair. This method, comprised of washing and cutting of hair, alkaline decomposition, hexane extraction, multilayer silica gel column chromatography, high performance liquid chromatography with a porous graphite carbon column and analysis by high resolution gas chromatography/high resolution mass spectrometry, enabled us to analyze PCDDs, PCDFs and Co-PCBs at trace levels of less than pg/g with good reproducibility. In addition, there was a correlation between some isomers in human hair and blood collected from identical donors. Human hair analysis is useful to evaluate human risk assessment including that due to environmental pollution.

Antibiotic effect of linolenic acid fromChlorococcum strain HS-101 andDunaliella primolecta on methicillin-resistantStaphylococcus aureus

Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains wasα-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of γ-linolenic acid.

Enhancement in fecal excretion of dioxin isomer in mice by several dietary fibers.

The effect of increased nutrients (protein, lipid, vitamins and minerals) on dioxin-induced toxic manifestations such as immune suppression, hepatic hypertrophy, splenic atrophy and enzyme induction was investigated in mice after oral administration of 1,2,3,4,7,8-HxCDD (HxCDD) as one of a representative compound of dioxin isomers. Consequently, it appeared that increased minerals and vitamins in the diet prevented immune suppression by HxCDD. In addition, to clarify the additive effect of nutrients and the ability to hasten the excretion of dioxins by dietary fiber, the adsorbing of dioxins by 16 dietary fibers was investigated by in vitro experiment. Among 16 dietary fibers, locust bean gum, pectin, alginic acid, guar gum, chitin and cellulose were effective in binding dioxin isomers. These dietary fibers also enhanced the fecal excretion of HxCDD in mice.

Anti-Herpes Simplex Virus substances produced by the marine green alga, Dunaliella primolecta

Among 106 microalgae tested, the cytopathic effect (CPE) upon Vero cells of herpes simplex virus, Type 1 (HSV-1) was inhibited by four methanol extracts of Dunaliella bioculata C-523, D. primolecta C-525, Lyngbya sp. M-9 and Lyngbya aerugineo-coerulea M-12. The green alga, D. primolecta, had the highest anti HSV-1 activity, since 10 μg mL-1 of extract from this alga completely inhibited the CPE. This activity was similar to that of acyclovir at the same concentration. We compared anti-viral activities against adeno virus, herpes simplex virus-2 (HSV-2), Japanese Encephalitis and Polio viruses. Only the CPE of HSV-2 was inhibited. Thus, the factor was specific against HSV. The antiviral activity was apparently excited during HSV adsorption and invasion of the cells. We optimized the conditions for anti HSV-1 activity by prolonging the exposure of HSV-1 to the extract. After 2 h, the CPE of even a high titer of HSV-1 (106 TCID50/0.1 mL) was completely inactivated. By use of various chromatographic techniques, three green substances having anti-HSV activity were purified from the algal mass of D. primolecta, and 5 μg mL-1 of this purified substances completely inhibited the CPE. From the analysis of NMR and MS, the chemical structures of the active substances were identified as pheophorbide-like compounds.

Formation of dioxin analogs by open-air incineration of waste wood and by fire of buildings and houses concerning Hanshin Great Earthquake in Japan.

Ash samples were collected from site of open-air incineration and fire concerning Hanshin Great Earthquake which occurred in western Japan in the early morning of 17 January 1995. The average concentrations of total sum of PCDDs, PCDFs and Co-PCBs in ash collected from wood scrap of broken building fire site, house fire site, shoe-making factory fire site and electric appliances store fire site were 541, 442, 859 and 22,800 pgTEQ/g, respectively. Particularly, PCDFs among dioxin analogs generated remarkably at sites with a high TEQ. In addition, 79,000 t of ash was totally produced at five open-air incineration sites, and consequently the formation amount of PCDDs, PCDFs and Co-PCBs was estimated to be 70.7 gTEQ on the basis of these analytical data.

Contamination levels of PCDDs, PCDFs and non-ortho chlorine substituted coplanar PCBS in milkfish and crab from culture pond and coastal area near open-air incineration sites for metal reclamation in Wan-li, Taiwan, Republic of China

Samples of culture freshwater milkfish (Chanos chanos) from five ponds surrounding open-air waste incineration sites and from five markets in southern Taiwan were analyzed for PCDDs, PCDFs and coplanar PCBs (Co-PCBs). The total TEQ concentration of three chemicals in fish meat was in a wide range of 0.195 to 153 pg/g wet weight. In addition, the bioconcentration factors from sediment to fish meat and crab hepatopancreas biota were arranged in order of Co-PCBs ⪢ PCDFs > PCDDs and Co-PCBs > PCDFs ⪢ PCDDs, respectively.

In vitro and in vivo induction of cytochrome P450 by coplanar polychlorinated/brominated biphenyls (Co-PXBs) providing high TEQ in mother's milk in Japan.

Coplanar polychlorinated/brominated biphenyls (Co-PXBs) are environmental pollutants previously identified in market fish samples. In this study, we observed that mothers milk in Japan is contaminated with Co-PXBs. Based on assumption that the toxicity of the same congener of PXDDs/DFs and Co-PXBs is nearly equal to that of the corresponding PCDDs/DFs and Co-PCBs, respectively, the toxic equivalent (TEQ) concentration was 10% of the total TEQ concentration (∑PCDDs/DFs, ∑PXDDs/DFs, ∑Co-PCBs and ∑Co-PXBs) in the milk. This observation suggested that humans, and especially infants, are exposed to high levels of Co-PXBs, which might cause adverse effects. However, the toxicity of Co-PXBs has to date not been reported. We assessed the toxic potency of Co-PXBs by studying their effect on the activity of cytochrome P450. Only the mRNA level and activity of CYP1A increased in a dose-dependent manner upon exposure to Co-PXBs. Substitution of bromine for chlorine into Co-PCBs provided higher CYP1A activity in in vitro and in vivo experiments. The expression level of aryl hydrocarbon receptor (AhR) mRNA was not altered, but luciferase activity, an indicator of AhR transcriptional activity, increased following treatment with Co-PXBs. The results suggest that CYP1A induction by Co-PXBs depended on AhR transcriptional activity and not on AhR expression. Although the TEFs of Co-PXBs are not set, if Co-PXBs are included in these calculations because of their higher toxicity compared to Co-PCBs, exposure to Co-PXBs cannot be neglected when assessing human health risks.

Property of cytochrome P450 1A inducibility by polychlorinated/brominated biphenyls (Co-PXBs) detected in Japanese breast milk

Coplanar polychlorinated/brominated biphenyls (Co-PXBs) belong to a class of structurally similar chemicals known as polyhalogenated aromatic hydrocarbons. We found that the milk of Japanese primiparous and multiparous mothers was similarly contaminated with Co-PXB congeners. Co-PXBs time- and dose-dependently increased ethoxyresorufin-O-deethoxylase (EROD activity) in HepG2 cells. The EROD activity of liver microsomes collected from C57BL/6 mice exposed to these congeners substituted with one or two, and with three or five bromine atoms time-dependently decreased and increased, respectively. These results indicate that introducing bromine into the chemical frame of a polychlorinated biphenyl tends to increase CYP1A activity in vitro and in vivo and that the number substituted bromine atoms alters the metabolism profiles. If Co-PXBs are more toxic than Co-PCBs, our findings suggest that the TEQ of Co-PXBs is important for human health risk.

Complex toxicity as disruption of adipocyte or osteoblast differentiation in human mesenchymal stem cells under the mixed condition of TBBPA and TCDD

People are frequently and unintentionally exposed to many chemical compounds, such as environmental pollutants and endocrine-disrupting chemicals (EDCs), in food and from the atmosphere. In particular, endocrine-disrupting TBBPA and dioxins are found in human breast milk and in the body. Conventional studies evaluate toxicity by administering a single substance to cells or animals, but evaluation of the toxicity of mixtures of these ingested compounds is essential for “true” toxicological assessment. We evaluated toxic effects in vitro using human mesenchymal stem cells (hMSCs). TBBPA increased the number of lipid droplets, and upregulated the expression of adipocyte-related mRNA, aP2 and LPL, through a PPARγ-dependent mechanism. TCDD suppressed lipid droplets and adipocyte-related mRNA levels. Adipocyte differentiation was stimulated by TBBPA and inhibited by TCDD in a dose-dependent manner. TBBPA did not influence osteoblast differentiation, but TCDD suppressed ALP staining and activity, calcium deposition, and osteoblast-related mRNA levels. In a mixture of TBBPA and TCDD, TBBPA inhibited TCDD suppression of adipocyte and osteoblast differentiation in a dose-dependent manner. Interestingly, we observed lipid droplets in TBBPA-treated cells differentiated into osteoblasts. These results suggest that TBBPA and TCDD disrupted differentiation into adipocytes and osteoblasts and contributes to a more complete toxicological understanding of exposure to these chemical substances.