Long-term cadmium exposure enhances metallothionein-1 induction after subsequent exposure to high concentrations of cadmium in P1798 mouse lymphosarcoma cells.

Cadmium, a ubiquitous heavy metal, is a toxic industrial and environmental pollutant. The initial biological response to cadmium exposure is induction of metallothioneins (MTs), a family of cysteine-rich, low-molecular-weight proteins that bind primarily zinc, cadmium, or both. This MT induction protects against cadmium toxicity by quenching cadmium. However, the effects of long-term cadmium exposure on MT1 gene expression are largely unknown. To investigate these effects, we used P1798 mouse lymphosarcoma cells, in which the MT1 gene is suppressed. As previously reported, MT1 expression remained unchanged after cadmium treatment. However, MT1 induction was observed in cells treated with 0.1 µM cadmium for 7 days, then exposed to 10 µM cadmium for 3 hr. In cells treated with 0.1 µM cadmium for 7 days, the transfected MT1 promoter reporter gene transcription and the cadmium incorporation in response to 10 µM cadmium induction were similar to those in untreated P1798 cells. Bisulfite genomic sequencing revealed that 7 day treatment with 0.1 µM cadmium slightly decreased CpG methylation in the 5´ flanking region of the MT1 gene. Our results together show that cadmium treatment results in MT1 induction and epigenetic modification of the MT1 gene.

Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk.

Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2',6-tribromobisphenol A (TriBBPA), 2,2'-dibromobisphenol A (2,2'-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPARγ transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary.

Levels of tetrabromobisphenol A, tribromobisphenol A, dibromobisphenol A, monobromobisphenol A, and bisphenol a in Japanese breast milk.

The levels of bisphenol A (BPA) and tetrabromobisphenol A (TeBBPA) were determined in breast milk samples from 19 Japanese mothers. BPA and TeBBPA levels were 36 ng/g lipid (range: 1.4-380 ng/g lipid) and 1.9 ng/g lipid (range: N.D. - 8.7 ng/g lipid), respectively. Tribromobisphenol A (TriBBPA) was similarly detected in all samples (mean: 5.5 ng/g lipid). We investigated the alteration of BPA-related compounds in breast milk over a period of three months. No trend could be observed for time-dependent changes in TeBBPA levels. High levels of TriBBPA were detected in breast milk samples with a high concentration of TeBBPA. We further examined concentration changes in BPA-related compounds in the breast milk of two donors over a period of 24 h. The results suggested that TriBBPA was a debrominated metabolite of TeBBPA, which had been ingested via food consumption and immediately transferred to the breast milk. On the basis of the present results, we estimated and compared the daily intake of BPA, TriBBPA, and TeBBPA from breast milk for infants. The estimated average intake of TriBBPA was 4 times higher than TeBBPA, at 48 and 12 ng/kg/day, respectively. The level of TeBBPA in breast milk was low, suggesting a low risk of causing adverse health effects. In conclusion, the concentration of both TriBBPA and TeBBPA must be determined in breast milk to accurately clarify the exposure of these compounds to infants.

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.

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 mother's 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.

Altered gene expression profiles associated with enhanced skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate in streptozotocin-diabetic mice.

To examine the mechanisms of diabetes-enhanced inflammation, ear inflammation was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in streptozotocin (STZ)-injected diabetic and control mice. The inflammatory response was determined from ear thickness and histology. The mRNA expression of several inflammation-related genes 8, 24 and 32 h after TPA treatment was determined by quantitative real-time RT-PCR. Ear thickness did not differ between the two groups at 8 h, but was greater in the diabetic mice than control mice at 24 and 32 h (late phase). STZ-diabetic conditions variously affected TPA-induced gene expression. The changes 8 h after TPA treatment probably reflected transcriptional regulation, and the genes were divided into three groups, up-regulated (IL-6, MCP-1, HO-1 and SOCS3), unregulated (IL-1beta, TNF-alpha and IL-10) and down-regulated (RANTES) genes. TPA-induced gene expression of cytokines, except for RANTES, peaked at 8 h and significantly declined in the late phase in control mice, while the expression of IL-1beta and TNF-alpha did not decline in the late phase in the diabetic mice. This result indicated the destabilization process for these mRNA, a type of post-transcriptional regulation, to be impaired under STZ-induced diabetic conditions; however, TPA-induced gene and protein expression of TTP, an RNA-binding protein involved in mRNA decay, were adversely enhanced in the diabetic mice. These findings suggested that STZ-induced diabetes affected the transcriptional and post-transcriptional control of TPA-induced inflammation, and greater mRNA levels of IL-1beta and TNF-alpha in the late phase were probably responsible for the diabetes-enhanced inflammation.

Efficacy of a bacterial siderophore, pyoverdine, to supply iron to Solanum lycopersicum plants.

Active uptake of ferric iron in microorganisms is based on siderophores. During iron deficiency, Pseudomonas fluorescens synthesizes siderophores, called pyoverdine, which have a high affinity for ferric iron. Strategy I plants generally cannot synthesize pyoverdine or take up ferric iron. We assessed the effect of pyoverdine chelated to ferric iron on iron nutrition in Solanum lycopersicum. Weight and photosynthetic pigment concentrations in the plants supplemented with the pyoverdine and ferric iron were restored to the rates of plants supplemented with ferrous iron. Leaves and roots accumulated significant iron after pyoverdine and ferric iron supplementation than when supplemented with ferric iron alone. When leaves and roots were supplemented with pyoverdine and ferric iron, the SlFRO1 expression level was suppressed to 20% and 50% relative to those decreased with ferric iron alone, respectively. The level of SlIRT1 in roots supplemented with pyoverdine and ferric iron decreased to 50% compared with the level in roots supplemented with ferric iron alone. These results suggest that SlFRO1 and SlIRT1 expression levels were suppressed and that iron content was restored by pyoverdine and ferric iron supplementation. Thus, the downregulation may have occurred because of negative feedback on mRNA expression. Pyoverdine-mediated ferric iron uptake by tomato is suggested to be a useful strategy to increase iron uptake from the environment.

[Investigation of PCB pollution evaluation for Yusho victims using their preserved umblical cord].

PCB and dioxin-like PCB (DL-PCB) in 28 umbilical cords preserved from the time when 3 Yusho victims and 11 healthy subjects gave birth were examined in order to investigate the pollution evaluation with both the compounds in Yusho victims on the basis of the analytical value. As a result, in a period of 1968 to the 70s immediately after Yusho incident, the total concentration of the 12 DL-PCB isomers in both the Yusho victims s and healthy subjects was very high. After that, the total DL-PCB concentration decreased temporally. However, there was not observed a significant difference in the concentration and composition of both contaminants between the two groups.

Global contamination of coplanar polybrominated/chlorinated biphenyls (Co-PXBs) in the market fishes from Japan.

Polybrominated chlorinated biphenyls (PXBs, X=Br, Cl) are a group of environmental contaminants that have not been studied previously. The introduction of the second halogen to the biphenyl backbone increases the number of possible congeners to more than 9000. Only a limited number of PXBs are commercially available. In order to determine the occurrence of these compounds in environmental matrices, an isotope dilution HRGC/HRMS method for determination of five co-planar polybrominated/chlorinated biphenyls (Co-PXBs) in biota was developed. The method detection limit for these compounds ranged between 0.05 and 0.5 pg/g for 4'-monobromo-3,3',4,5-tetrachlorobiphenyl and 3',4',5'-tribromo-3,4-dichlorobiphenyl, respectively. Concentrations of five co-planar polybrominated and chlorinated biphenyls in eighteen different fish fillets from Japanese markets ranged between 4 and 46 pg/g wet weight for mink whale and young yellow-tail fish. These values are substantially lower than those reported for Co-PCBs; however, it should be noted that due to the unavailability of standards, identification and quantification of all the isomers was not possible.

Analytical method for determination of dinitropyrenes using gas chromatography-high-resolution mass spectrometry.

A gas chromatography-high-resolution mass spectrometry method has been developed for the determination of dinitropyrenes (DNPs). DNPs were reduced to the corresponding diaminopyrenes with sodium hydrosulfide, and the reduced DNPs were derivatized with N-methyl-bis(trifluoroacetamide). The sensitivity of this method was up to 10 times that of high-performance liquid chromatography with fluorescence detection. The detection limits for 1,3-, 1,6-, and 1,8-DNPs were in the 0.90-1.1 pg range. The concentrations were corrected with a correction factor determined from the recovery of an internal standard. The recovery of deuterium-labeled 6-nitrochrysene was at least 87%. This new method was utilized for quantitative determination of DNPs in Standard Reference Material (SRM) 1975 available from the US National Institute of Standards and Technology. Three DNP isomers were measured in SRM 1975 at 0.571-1.59 microg/g.

Formation of toxic chemicals including dioxin-related compounds by combustion from a small home waste incinerator.

We investigated combustion in a small home waste incinerator and analyzed both flue gas and residual ash for formation of the dioxin-related compounds polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls and their precursors polychlorinated benzenes, polychlorinated phenols, polychlorinated diphenyl ethers, and polychlorinated biphenyls. Particularly, we investigated the effect of the incinerated material's composition on both the congener ratios of released compounds and the total concentration of all congeners of each compound. Eight different samples were prepared for incineration with four samples consist entirely paper, dead leaves, natural wood, or building materials. The remaining four samples contained mostly paper, but also other components such as fiber, non-chlorine-containing plastics, chlorine-containing plastics, and copper electric wire. The presence of non-chlorine-containing plastic in combustion samples did not increase overall dioxin or dioxin-precursor emissions. In contrast, chlorine-containing plastic resulted in a several-fold increase in total polychlorinated dioxins released, in both flue gas and residual ash. Copper wire resulted in a further several-fold increase in total polychlorinated dioxins and dioxin precursors released, with one exception: the addition of chlorine-containing plastic resulted in a many-fold increase in polychlorinated biphenyls, but only a modest further increase ( approximately 52%) with the further addition of copper. Homologue ratios tended toward higher-chlorine compounds as chlorine-containing plastic and Cu were added, but the results were far from uniform. Our results show that toxic dioxin release from small home waste incinerators must be considered significant, especially if even small amounts of chlorine-containing plastics or copper are burned.

Large-scale evaluation of the current level of polybrominated diphenyl ethers (PBDEs) in breast milk from 13 regions of Japan.

Polybrominated diphenyl ethers (PBDEs) were measured in 2004 in 105 breast milk samples collected from 13 regions of Japan (Hokkaido, Akita, Miyagi, Tokyo, Gifu, Fukui, Kyoto, Hyogo, Wakayama, Shimane, Yamaguchi, Kochi and Okinawa). Six congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154) were determined by gas chromatography /mass spectrometry (GC/MS). Total PBDE levels ranged from 0.01 to 23.0 ng/g lipid (geometric mean (GM), 1.34 ng/g lipid). BDE-47 (GM, 0.66 ng/g lipid, 59% of sigmaPBDE) was the most abundant congener present in breast milk and was detected in 99% of the samples. Total PBDE levels were higher in northern Japan than in other regions. We analyzed the effects of occupation, age, smoking status, alcohol consumption and number of deliveries on total PBDE levels. None of these factors were significantly associated with the level of PBDEs. The present study revealed that the current level of exposure to PBDEs in Japan is lower than that in the USA or Sweden. GMs (ng/g lipid) (GSD, geometric standard deviation) and medians (ng/g lipid) of PBDE levels in each district are as follows: Hokkaido 2.70 (1.70), 2.74; Akita 4.49 (2.19), 5.44; Miyagi 1.77 (4.37), 1.11; Tokyo 1.39 (2.09), 1.63, Gifu 2.83 (4.79), 2.23; Fukui 1.05 (2.34), 1.18; Kyoto 1.31 (2.95), 1.33; Hyogo 1.02 (2.69), 0.88; Wakayama 1.33 (3.80), 1.70; Shimane 0.83 (2.51), 0.66; Yamaguchi 1.74 (2.82), 1.76; Kochi 0.50 (2.69), 0.74 and Okinawa 1.91 (2.75), 1.22. This is the first large-scale study of current PBDE levels in breast milk in Japan.

Optimization of the purification method for dioxin analysis in human serum and temporal changes in background dioxin levels in the general population.

To perform high quality and high throughput measurements, complicated purification procedure by two rounds of open column chromatography was simplified by appraising the components and adding a sufficient amount of adsorbents to the column. The procedure consists of two open column techniques using a multi-layer silica gel column and an active carbon dispersed silica gel column. This purification procedure is used in "Standard manual for dioxin analysis in human blood" published in 2000 (Ministry of Health and Welfare, Japan, 2000). Consequently, a purification procedure using only 1 g of 10% AgNO(3)/silica gel can remove the matrix of blood serum as effectively as a multi-layer silica gel column. In addition, 0.1 g of carbon silica gel, equivalent to 1/10 the weight used currently in analyses, separates and recovers the dioxins in purified extract of blood serum. Furthermore, changes in the background level in the general population were clarified by monitoring the dioxin concentrations in human blood. The dioxin concentrations and ratios in blood in the general population remained at a tolerable level for three months. Therefore, the blood serum was available for the detection of a remarkable elevation in dioxins level considered to be indicative of contamination due to high-density exposure.

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.

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.

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.