Distributions and multiple sources of chlorinated polycyclic aromatic hydrocarbons in the air over Japan.

Chlorinated polycyclic aromatic hydrocarbons (ClPAHs) have been detected in the air at discrete sites in Japan, but there is no information on their distributions throughout Japan. This study was a widespread survey of atmospheric concentrations of ClPAHs throughout Japan. The majority of 24 species of ClPAHs were detected in either the gas or particle phase at all sampling sites. The concentrations were weakly related to human population densities. The relationships between total concentrations of ClPAHs and PAHs suggested that atmospheric ClPAHs at ~70% of the sites were derived from common sources of PAHs. A ternary diagram based on diagnostic ratios of 1-chloropyrene, 7-chlorobenz[a]anthracene, and 6-chlorobenzo[a]pyrene normalized to 3-chlorofluoranthene suggested that waste combustion was the likely source of ClPAHs in summer and that vehicular emissions and coal burning were the likely sources of ClPAHs in winter. A heat map analysis estimated from the concentrations of individual compounds at each site suggested that there were three categories of sources at the sites: i) common sources of ClPAHs and PAHs that had moderate impacts, ii) common sources of ClPAHs and PAHs that had high impacts, and iii) specific sources of ClPAHs.

Detection and measurement of the agonistic activities of PCBs and mono-hydroxylated PCBs to the constitutive androstane receptor using a recombinant yeast assay.

Polychlorinated biphenyls (PCBs) are thought to exert their toxicities mainly by binding to the aryl hydrocarbon receptor and by stimulating transcription of various genes, notably metabolizing enzymes including the cytochrome P450 (CYP) 1 family. However, PCBs and their metabolites could have potential to activate other nuclear receptors and subsequent events. We focused on the constitutive androstane receptor (CAR) inducing CYP2B and measured the agonistic activity of PCBs and mono-hydroxylated PCBs (OH-PCBs) to the CAR using yeast cells transduced with the human CAR and its response pathway. Twenty-nine of 34 tested PCBs and 72 of 91 OH-PCBs exhibited CAR agonistic effects. Of 41 OH-PCBs that had the same chlorination patterns as the tested PCBs, 9 had activities more than twice those of their non-hydroxylated analogs. In particular, 2',4',6'-trichlorobiphenyl-4-ol and 2,2',4',6'-tetrachlorobiphenyl-4-ol were 332- and 22-fold more potent than their analogs and were 15 times and 2.8 times, respectively, as active as a reference substance, 4-tert-octylphenol. The activities of 17 of the OH-PCBs were reduced to less than half those of their non-hydroxylated analogs. Four OH-PCBs derived from 3 active PCBs were inactive. However, a consistent relationship between hydroxyl substituent position and activity could not be discerned. Comprehensive evaluation of the toxic potential of PCBs and their hydroxylated metabolites and their concentrations in the environment are required.

Effects of transovarian exposure to p,p'-DDT and p,p'-DDE on avian reproduction using Japanese quails.

In the 1950s to 1970s developed countries reported declines in populations of raptorial and fish-eating birds and dichlorodiphenyltrichloroethane (DDT) and its metabolites were considered causative substances because they accumulated significantly in the tissues of wild birds and animals. However, except for the estrogenic effects of o,p'-DDT, a minor component of commercial DDT, there has been no compelling evidence that DDT directly affects avian reproductive systems. To assess the possible impact of DDT on development and reproduction of birds, exposure experiments to the major component of commercial DDT, p,p'-DDT, and its persistent metabolite, p,p'-dichlorodiphenyldichloroethylene (DDE), were performed using Japanese quail (Coturnix japonica) eggs; the test substances (3 to 100 µg/g) were injected into the yolk prior to incubation, and hatched chicks were raised to adulthood. p,p'-DDT had no significant effects on the morphology and function of the reproductive systems, although the hatchability of treated eggs was reduced at the highest dose (100 µg/g). High doses of p,p'-DDE slightly enhanced the eggshell forming ability of female quails; eggshell mass and thickness were increased at 30 µg/g or more although no morphological changes were observed in the oviduct. Transcriptions of the CYP11A1 gene in the ovaries, and of AHR and ARNT in the livers, of adult females were significantly increased at 3 µg/g or more of p,p'-DDT. Except for low hatchability, transovarian exposure to p,p'-DDT or p,p'-DDE did not markedly impair the avian reproductive systems, but the hormonal actions of these compounds are likely to change reproductive and hepatic functions even after maturation.

Characteristics of the transformation frequency at the tumor promotion stage of airborne particulate and gaseous matter at ten sites in Japan.

We used a high-volume air sampler in the summer of 2007 and the winter of 2008 at ten Japanese sites (Sapporo, Sendai, Maebashi, Tsukuba, Shinjuku, Sagamihara, Shizuoka, Touhaku, Kitakyushu, and Kagoshima) to collect total suspended particulate (TSP) and gaseous matter for evaluation. We evaluated the transformation frequency at the tumor promotion stage of these samples in a cell transformation assay using Bhas 42 cells, which were established from BALB/c 3T3 cells transfected with the v-Ha-ras oncogene. All samples collected from the gaseous matter were negative for transformed foci. There were several patterns of transformation frequency at the tumor promotion stage by area for the TSP samples. At Sapporo, the transformation frequency at the tumor promotion stage was remarkably higher in winter than in summer as well as in winter at the other sites. At six urban cities from Sendai to Shizuoka, the levels of transformed frequencies per µg of suspended particulates in winter were almost the same, and were higher than those of the remaining three sites. At three sites, Touhaku, Kitakyushu and Kagoshima, the transformation results in winter were judged as negative. The characteristics of the transformed frequencies of the compounds adsorbed on particulate matter at the sampling sites were significant in winter. We also studied the correlation between the transformation frequency at the tumor promotion stage of the TSP samples and the results of quantitative analysis of 16 polyaromatic hydrocarbons (PAHs) at the ten sites. We found that the transformation frequency at the tumor promotion stage of airborne samples could not be predicted based on the quantitative results of the PAHs in those samples. These data suggest that direct risk assessment of air samples with a bioassay is more valuable than quantitative analysis of compounds such as PAHs for predicting carcinogenicity.

A pilot survey of 39 Victorian WWTP effluents using a high speed luminescent umu test in conjunction with a novel GC-MS-database technique for automatic identification of micropollutants.

In 2007, samples of treated effluent were collected at point of discharge to the environment from 39 wastewater treatment plants (WWTPs) located across Victoria, Australia grouped by treatment type. Sample genotoxicity was assessed with a high-throughput luminescent umu test method using Salmonella typhimurium TL210 strain, with and without addition of a commercially available metabolic activation system. Samples were also screened using a gas chromatographic-mass spectrometric mass-structure database recognition method. A genotoxic response was observed in half of the samples tested without metabolic activation system (

A pilot study of the water quality of the Yarra River, Victoria, Australia, using in vitro techniques.

A pilot study was initiated to provide the first information on the recombinant receptor-reporter gene bioassay (hormonal) activity of freshwaters in Victoria. The project involved the collection of water samples from six stations on the main stem of the Yarra River in and upstream of the city of Melbourne, Australia in April 2008 and April 2009. Samples were prepared for measurement of sample toxicity using a modified photobacterium test, genotoxicity using a high-throughput luminescent umu test method, and human and medaka estrogen receptor (hERα and medERα), retinoic acid receptor (RAR), aryl hydrocarbon receptor (AhR) and thyroid receptor (TR) assay activity using the relevant yeast-based bioassays. Most samples were only weakly or moderately toxic, with no relationship observed to location along the river. The data for 2008 suggests that at that time the Yarra River samples contained few compounds that were, in and of themselves, genotoxic. No estrogenic or thyroid, and <1 ng/L retinoic acid receptor activity was observed. AhR activity increased with progressed downstream. AhR activity was higher in April 2009 than at the same time in 2008, perhaps as a result of extensive bush fires in the catchment in the months immediately prior to sampling. About 24% of the total AhR activity observed was associated with suspended solids.

The feasibility of using mosquitofish (Gambusia affinis) for detecting endocrine-disrupting chemicals in the freshwater environment.

We evaluated the utility of gene-transcriptional responses in the liver of mosquitofish (Gambusia affinis), a species introduced to many countries and therefore widely available, for detecting endocrine-disrupting activity in water. Exposure to ß-naphthoflavone, an aryl hydrocarbon receptor (AhR) agonist, significantly increased the transcript of the cytochrome P4501A gene (cyp1a), peaking at 24 h, in both sexes at concentrations of 10 µg/L or more. 17ß-Estradiol (E(2) ) at 500 ng/L increased the number of males showing gene transcription of precursors of yolk protein, vitellogenin (Vtga, Vtgb, and Vtgc), at 24, 48, and 72 h. Exposure for 48 h to bisphenol A (BPA), an estrogen mimic, also increased vtg-positive males at 1 mg/L or more. Leachate from a Japanese stable-type landfill significantly increased vtg-positive males after 48 h exposure, and the in vitro activity of the leachate against the estrogen receptor (ER) was estimated as an E(2) equivalent of 240 ng/L by yeast transfected with the ER. Chemical analysis showed that major contributors to the ER activation were BPA and 4-tert-octylphenol. This leachate and drainage water from a control-type landfill had AhR activities, estimated by yeast with the AhR, but had no significant effect on cyp1a transcription. These results showed that mosquitofish are suitable for detecting in vivo AhR and ER effects, but are insensitive to E(2).

Estrogenic effects of leachates from industrial waste landfills measured by a recombinant yeast assay and transcriptional analysis in Japanese medaka.

In Japan, the leachates from 'stable type' landfills for industrial wastes are not controlled, and this has given rise to concerns about the possible pollution of surrounding environmental waters, especially by endocrine disrupting chemicals leaching from plastic and rubber wastes. To accurately assess the estrogenic potential of the landfill leachates by both in vitro and in vivo approaches, we confirmed gene-transcriptional responses in recombinant yeast cells and in Japanese medaka fish to estrogenic compounds, and applied these transcription assays to leachate samples. The yeast carrying the estrogen receptor (ER) of medaka and an ER-mediated response pathway responded to both the natural estrogen, 17ß-estradiol (E2), and an industrial compound, bisphenol A (BPA), and the effective concentration of BPA was about 2.0×10(3) times that of E2. Transcripts of all genes coding for precursors of yolk protein, vitellogenin (vtg1 and vtg2), and precursors of egg envelope subunit proteins, choriogenins (chgh and chgl), increased in a concentration dependent manner in the livers of male medaka exposed to BPA or E2, and, except for chgh, reached peaks at exposure times of 48h. Although many fish in control groups did not have vtg transcripts, the incidence of vtg transcriptions also increased in a concentration dependent manner with exposure. The minimum effective concentrations of BPA at 48h were 0.5mg/L for chgh and vtg2, 2mg/L for vtg1 and 4mg/L for chgl, while those of E2 were 10ng/L for chgh and chgl and 30ng/L for vtg1 and vtg2. All leachates sampled at 3 landfill sites exerted in vitro estrogenic action. The E2 equivalent of the most potent leachate was 375ng/L for the yeast ER assay. This leachate sample significantly increased the transcripts of chgh, vtg1 and vtg2, but not chgl, in the medaka. In addition, chemical analysis showed that bisphenol A, 4-tert-octylphenol and 4-nonylphenol were the main contributors to the estrogenicity of the leachates. This study indicated that this type of landfill may adversely affect the reproductive functions of fish living in the surrounding area by leakage of industrial estrogenic compounds.

Effect of D-cycloserine on spatial learning performance and memory function-related gene expression in mice following toluene exposure.

D-cycloserine (DCS), a partial N-methyl-D-aspartate (NMDA) receptor agonist, is a well-known cognitive enhancer. To investigate the effect of DCS in cognitive function following toluene exposure, eight-week-old male C3H/HeN mice were exposed to filtered air (0 ppm) or 50 ppm toluene for 6 h a day on 5 consecutive days a week for 6 weeks. The day after the day of final exposure, a spatial learning task was performed using a Morris water maze apparatus. During the learning task, some mice were treated with DCS intraperitoneally (20 mg/kg) 30 min before the first trial of the acquisition phase and probe trial. After completion of the spatial learning task, the hippocampus was collected from each mouse to examine memory function-related gene expression using the real-time RT-PCR method. During the acquisition phase, on day 3 and 4, toluene-exposed mice with DCS treatment showed significantly better learning performance than corresponding saline treated groups. Moreover, the toluene-exposed mice with DCS treatment also showed significantly improved memory retention during the probe trial and up-regulation of hippocampal NMDA receptor subunit 2B mRNA expression compared with the saline treated groups. Our findings indicate that a subunit-specific modulation of hippocampal NMDA receptor mRNA expression by DCS contributes to improvement of spatial learning performance in mice following toluene exposure.

Mutagenicity and PAH contents of soil in forests or planted areas in Japan.

We investigated the behavior of mutagenic substances in the soil of forests or planted areas. Mutagenicity and concentration was examined for 16 types of PAHs in soil samples collected at a depth of 1 m in 10 forests in Iwate, Ibaraki, Tokyo, Kanagawa, Yamanashi and Shizuoka prefectures in Japan. Mutagenicity and PAHs were detected mostly in soil from the surface to a depth of 30 cm when strains TA100, TA98 and YG1024 were used. In addition, a significant correlation was not found between the concentration of BaP, and specific mutagenic activity (TA98 without S9mix, r = 0.285).

Effects of maternal toluene exposure on testosterone levels in fetal rats.

The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD 18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17alpha-hydroxylase/c17-20 lyase, 17beta-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3beta-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 3beta-HSD expression.

Activity related to the carcinogenicity of plastic additives in the benzophenone group.

This study examines the activities relating to the carcinogenicity of six types of benzophenone derivatives (benzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2,4-dihydroxybenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone and 2,2'-dihydroxy-4,4'-dimethoxybenzophenone) currently used in plastic products as additives to serve as ultraviolet absorbing agents. The evaluation of the initiation activity used a light absorption umu-test, a luminescent umu-test and the Ames test. The promotion activity was examined by a Bhas assay, a method that uses Bhas 42 cells for the formation of transformation foci. The luminescent umu-test indicated positive initiation activity of 2-hydroxy-4-methoxybenzophenone, and pseudo-positive activity of 2,4-dihydroxybenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone. In the Ames test, 2-hydroxy-4-octyloxybenzophenone showed pseudo-positive initiation activity. Conversely, 2,4-dihydroxybenzophenone indicated weak promotion activity at 10 microg/ml concentration.