Historical and geographical aspects of the increasing perfluorooctanoate and perfluorooctane sulfonate contamination in human serum in Japan.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) have recently received attention due to their widespread contamination in the environment, as well as in wildlife and humans. We measured the PFOS and PFOA concentrations in historically recorded human serum samples at an age range between 20 and 59 years collected in Kyoto, 20 persons per each time point (n=100), and also the PFOS and PFOA concentrations in human serum samples at an age range between 20 and 59 years from 10 locations throughout Japan (n=200). The historical samples collected from 1983 to 1999 demonstrated that the PFOA concentrations in males and females from Kyoto have increased 4.4-fold and 4.3-fold at a rate of increase of 0.49 ng/ml/year and 0.42 ng/ml/year, respectively. In contrast, serum concentrations of PFOS reached a plateau in the late 1980s. There are also regional differences in both the PFOS and PFOA serum concentrations. The concentrations in serum [geometric mean (geometric standard deviation)] (ng/ml) in 2003-2004 ranged from 7.6(1.6) in the town of Matsuoka in Fukui prefecture to 27.8(1.6) in Kyoto city, and ranged from 2.3(1.5) in Matsuoka to 14.5(1.3) in Osaka city for PFOS and PFOA, respectively.

Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis. The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution. The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate. The crystal structure of E. coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution. Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site. On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

Endoplasmic reticulum stress induces Wfs1 gene expression in pancreatic beta-cells via transcriptional activation.

OBJECTIVE: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic beta-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. DESIGN AND METHODS: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic beta-cell-derived MIN6 cells, and Akita mouse-derived Ins2 (96Y/Y) insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. RESULT: Islet beta-cells were the major site of Wfs1 expression. This expression was also found in delta-cells, but not in alpha-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 (96Y/Y) insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 (96Y/Y) cells than Ins2 (wild/wild) cells. CONCLUSION: Wfs1 expression, which is localized to beta- and delta-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

Secular trends and geographical variations in the dietary intake of polybrominated diphenyl ethers (PBDEs) using archived samples from the early 1980s and mid 1990s in Japan.

A retrospective exposure assessment among the general population for polybrominated diphenyl ethers (PBDEs) was conducted using dietary surveys. We analyzed samples of food duplicate portions collected in the early 1980s (1980 survey: N=40) and the mid 1990s (1995 survey: N=39) from female subjects (5 participants from each of 8 sites per survey except for one site) living throughout Japan, from the north (Hokkaido) to the south (Okinawa). The study populations in the 1980 and 1995 surveys were different, but lived in the same communities. We measured four PBDE congeners [2,2',4,4'-tetrabrominated diphenyl ether (tetraBDE): #47; 2,2',4,4',5-pentaBDE: #99; 2,2',4,4',6-pentaBDE: #100; and 2,2',4,4',5,5'-hexaBDE: #153] in the diet. #99 was the most abundant congener in the diet (49% of the total PBDEs), followed by #47 (33%), #100 (12%) and #153 (6%). Regional variations found in the 1980 survey decreased in the 1995 survey. The total daily intake of PBDEs (ng/d) [GM (GSD)] in the 1980 survey [91.4 (4.1)] was not significantly different from that in the 1995 survey [93.8 (3.4)] for the total population, nor did it differ among the sites including Shimane, in which a 20-fold increase in serum concentrations was observed in the same population1). In consideration of the significant increases in the serum concentration, inhalation may be more important than food ingestion as the route of human exposure to PBDEs.

Mutation analysis in PKD1 of Japanese autosomal dominant polycystic kidney disease patients.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder (incidence, 1:1,000). The mutation of PKD1 is thought to account for 85% of ADPKD. Although a considerable number of studies on PKD1 mutation have been published recently, most of them concern Caucasian ADPKD patients. In the present study, we examined PKD1 mutations in Japanese ADPKD patients. Long-range polymerase chain reaction (LR-PCR) with PKD1-specific primers followed by nested PCR was used to analyze the duplicated region of PKD1. Six novel chain-terminating mutations were detected: three nonsense mutations (Q2014X transition in exon 15, Q2969X in exon 24, and E2810X in exon 23), two deletions (2132del29 in exon10 and 7024delAC in exon 15), and one splicing mutation (IVS21-2delAG). There was also one nonconservative missense mutation (T2083I). Two other potentially pathogenic missense mutations (G2814R and L2816P) were on the downstream site of one nonsense mutation. These three mutations and a following polymorphism (8662C>T) were probably the result of gene conversion from one of the homologous genes to PKD1. Six other polymorphisms were found. Most PKD1 mutations in Japanese ADPKD patients were novel and definitely pathogenic. One pedigree did not link to either PKD1 or PKD2.

Absence of linkage of familial intracranial aneurysms to 7q11 in highly aggregated Japanese families.

BACKGROUND AND PURPOSE: We sought to test the linkage of familial intracranial aneurysms (FIAs) to the ELN (elastin) locus in chromosome 7q11 reported previously. METHODS: Intracranial aneurysm (IA) probands were searched from patient records or neurosurgeons' recalls in collaborating hospitals. Members of the participating probands' families who had unknown affection status were screened by MR angiography and diagnosed by digital subtraction angiography. Inclusion criteria of families for genetic analyses were as follows: at least 3 alive affected members or 2 alive affected members with at least 1 unaffected member (>or=60 years). Linkage to the ELN locus was tested with the use of GENEHUNTER by parametric and nonparametric methods. To exclude false-negatives in the linkage analysis, the lowest 5% limits of logarithms of the odds (LOD) and nonparametric LOD (NPL) scores for individual families and for the total set of families were simulated on assumption that the ELN locus is linked to FIAs. RESULTS: Questionnaires were sent to 885 patients, and 563 responded. Seventy-nine probands were positive for family history. One hundred thirty-four family members of unknown affection status were screened. A total of 14 families with 64 members met the criteria. Linkage to the ELN locus was discarded in 11 families and was inconclusive for 3 families. The total LOD and total NPL scores for 14 families were -8.04 and -0.643, respectively. Our conclusion did not change even when the values of penetrance were changed or only affected members were analyzed. CONCLUSIONS: The majority of aggregated IA Japanese families may not have a genetic linkage to chromosome 7q11.

The influence of time, sex and geographic factors on levels of perfluorooctane sulfonate and perfluorooctanoate in human serum over the last 25 years.

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are important perfluorochemicals (PFCs) in various applications. Recently, it has been shown that these chemicals are widespread in the environment, wildlife and humans. But the kinds of factors that affect their levels in serum are unclear, and it is also not clear whether exposure to them is increasing or not. To investigate the impacts of time, geographical location and sex on the levels of these chemicals, we measured PFOS and PFOA concentrations in human sera samples collected both historically and recently in Miyagi, Akita and Kyoto Prefectures in Japan. The PFOS and PFOA levels in sera [Geometric Mean (Geometric Standard Deviation)] (microg/L) in 2003 ranged from 3.5 (2.9) in Miyagi to 28.1 (1.5) in Kyoto for PFOS and from 2.8 (1.5) to 12.4 (1.4) for PFOA. Historical samples collected from females demonstrated that PFOS and PFOA concentrations have increased by factors of 3 and 14, respectively, over the past 25 yr. There are large sex differences in PFOS and PFOA concentrations in serum at all locations. Furthermore, there are predominant regional differences for both PFOS and PFOA concentrations. In Kyoto the concentrations of PFOA in dwellers who had lived in the Kinki area for more than 2 yr were significantly higher than in people who had recently moved into the area, in both sexes. This finding suggests that there are sources of PFOA in the Kinki area that have raised the PFOA serum levels of its inhabitants. Further studies are needed to elucidate these sources in the Kinki area of Japan.

Perfluorooctanoate and perfluorooctane sulfonate concentrations in surface water in Japan.

Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are synthetic surfactants used in Japan. An epidemiological study of workers exposed to PFOA revealed a significant increase in prostate cancer mortality. A cross-sectional study of PFOA-exposed workers showed that PFOA perturbs sex hormone homeostasis. We analyzed their concentrations in surface water samples collected from all over Japan by LC/MS with a solid phase extraction method. The lowest limits of detection (LOD) (ng/L) were 0.06 for PFOA and 0.04 for PFOS. The lowest limits of quantification (LOQ) (ng/L) were 0.1 for both analytes. The levels [geometric mean (GM); geometric standard deviation (GS)] (ng/L) of PFOA and PFOS in the surface waters were GM (GS): 0.97 (3.06) and 1.19 (2.44) for Hokkaido-Tohoku (n=16); 2.84(3.56) and 3.69 (3.93) for Kanto (n=14); 2.50 (2.23) and 1.07 (2.36) for Chubu (n=17); 21.5 (2.28) and 5.73 (3.61) for Kinki (n=8); 1.51 (2.28) and 1.00 (3.42) for Chugoku (n=9); 1.93 (2.40) and 0.89 (3.09) for Kyushu-Shikoku (n=15). The GM of PFOA in Kinki was significantly higher than in other areas (ANOVA p<0.01). Systematic searches of Yodo and Kanzaki Rivers revealed two highly contaminated sites, a public-water-disposal site for PFOA and an airport for PFOS. The former was estimated to release 18 kg of PFOA/d. PFOA in drinking water in Osaka city [40 (1.07) ng/L] was significantly higher than in other areas. The present study confirms that recognizable amounts of PFOA are released in the Osaka area and that people are exposed to PFOA through drinking water ingestion.

[The legal issue of the present occupational physician system in crisis management of health damage].

In present day Japan, when a crisis like the Bhopal accident occurs, due to defects in the current of industrial health law, effective crisis management cannot be taken to minimize health damage in both workers and residents. The current law characterizes industrial health as a part of the welfare service provided by employers for employees. Nevertheless, the company should be liable for all failure and damage including health impairment as a primary party. Moreover, in an emergency crisis, unlike the case of reparations, it is not accepted to argue whether the company should take absolute liability or not. Accordingly, in such cases, we consider it more appropriate to apply the "Polluter Pays Principle" and the principle of "Liability without Fault" to the company's responsibility. By these rules, the company should mobilize their own professionals, who are experts of managing crises, such as occupational physicians and/or industrial health professionals to minimize health damages among citizens in general. The company should take such a social responsibility in a crisis when it is the primary responsible party to the crisis.

Reconstruction of human exposure to heavy metals using synchrotron radiation microbeams in prehistoric and modern humans.

OBJECTIVE: Teeth can serve as records of environmental exposure to heavy metals during their formation. We applied a new technology - synchrotron radiation microbeams (SRXRF) - for analysis of heavy metals in human permanent teeth in modern and historical samples. METHODS: Each tooth was cut in half. A longitudinal section 200 mum in thickness was subjected to the determination of the heavy metal content by SRXRF or conventional analytical methods (ICP-MS analysis or reduction-aeration atomic absorption spectrometry). The relative concentrations of Pb, Hg, Cu and Zn measured by SRXRF were translated in concentrations (in g of heavy metal/g of enamel) using calibration curves by the two analytical methods. RESULTS: Concentrations in teeth in the modern females (n = 5) were 1.2 +/- 0.5 mug/g (n = 5) for Pb; 1.7 +/- 0.2 ng/g for Hg; 0.9 +/- 1.1 mug/g for Cu; 150 +/- 24.6 mug/g for Zn. The levels of Pb were highest in the teeth samples obtained from the humans of the Edo era (1603-1868 AD: ) (0.5-4.0 mug/g, n = 4). No trend was observed in this study in the Hg content in teeth during 3,000 years. The concentrations of Cu were highest in teeth of two medieval craftsmen (57.0 and 220 mug/g). The levels of Zn were higher in modern subjects (P < 0.05) than those in the Jomon (~1000 BC: ) to Edo periods [113.2 +/- 27.4 (mug/g, n = 11)]. Reconstruction of developmental exposure history to lead in a famous court painter of the Edo period (18th century) revealed high levels of Pb (7.1-22.0 mug/g) in his childhood. CONCLUSIONS: SRXRF is useful a method for reconstructing human exposures in very long trends.

The bioconcentration factor of perfluorooctane sulfonate is significantly larger than that of perfluorooctanoate in wild turtles (Trachemys scripta elegans and Chinemys reevesii): an Ai river ecological study in Japan.

Turtles rank high in the river food chain, and are suitable for predicting the bioconcentrations of chemicals through the food chain. Trachemys scripta elegans (N=46) and Chinemys reevesii (N=51) were captured in a river in Japan, from September to October 2003 and April to June 2004. Surface water samples were collected simultaneously from the same sites at which the turtles were caught. Serum perfluorooctane sulfonate (PFOS) ranged from 2.4 to 486 microg/L, while water PFOS levels ranged from 2.9 to 37 ng/L. The geometric mean (GM) (geometric standard deviation, GSD) of the bioconcentration factor (BCF) of PFOS was 10,964 (2.5). In contrast, the perfluorooctanoate (PFOA) level in water ranged from 16.7-87,100 ng/L, and serum PFOA ranged from <0.2 to 870 microg/L. The GM (GSD) of the BCF of PFOA was 3.2 (7.9). Furthermore, the BCF of PFOA decreased as the PFOA level in the surface water increased. PFOS could be preferentially bioconcentrated in biota, and PFOA, slightly bioconcentrated.

Renal clearance of perfluorooctane sulfonate and perfluorooctanoate in humans and their species-specific excretion.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are detected in the environment, as well as more specifically in wildlife and humans. However, the toxicokinetic aspects of perfluorochemicals in humans are unclear. In this study, we measured concentrations of PFOA and PFOS in subjects who had lived in Kyoto city for more than 10 years. The serum concentrations of PFOA and PFOS were higher in females who menstruated than those who did not menstruation (P<0.01), but in males this did not change by age; the levels in females reached those in males at an age of 60 years. We then determined the renal clearances of PFOA and PFOS in young (20-40 years old, N=5 for each sex) and old (60 years old, N=5 for each sex) subjects of both sexes. All young females were menstruating, while all old females were not. The renal clearances were 10(-5)-fold smaller than the glomerular filtration rate in humans, suggesting the absence of active excretion in human kidneys. The renal clearances of PFOA and PFOS were approximately one-fifth of the total clearance based on their serum half-lives, assuming a one-compartment model. The sex differences in renal clearance that have been reported in rats and Japanese macaques were not found in our human subjects. We tried to build a one-compartment pharmacokinetic model using the reported half-lives in human. The model was simple but could predict the serum concentrations in both males and females fairly well. We therefore suggest that an internal dose approach using a pharmacokinetic model should be taken because of the large species differences in kinetics that exist for PFOA and PFOS.

Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface.

A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.

Assessment of human exposure to polychlorinated biphenyls and polybrominated diphenyl ethers in Japan using archived samples from the early 1980s and mid-1990s.

Persistent organic pollutants have been linked to various adverse effects on human health. We conducted a retrospective exposure assessment for 11polychlorinated biphenyl (PCB) congeners and 4 polybrominated diphenyl ether (PBDE) congeners. We analyzed paired samples of blood and food duplicate portions collected in the 1980s (1980 survey, N=40) and the mid-1990s (1995 survey, N=40) from females (five participants from each of eight sites per survey) living throughout Japan, from Hokkaido to Okinawa. The study populations in the 1980 and 1995 surveys were different but had lived in the same community. We measured PCBs and PBDEs in serum and PCBs in diet. Total serum PCBs (ng/g lipid) [geometric mean (geometric standard deviation)] were similar in the 1980 [163.0 (1.7)] and the 1995 [142.6 (2.0)] surveys. In contrast, dietary intake (ng/day) between 1980 and 1995 decreased significantly, from 522.8 (2.5) to 165.9 (3.3), respectively, (P<0.05). We classified the participants by birth year-before 1941 (older generation) and equal to or after 1941 (younger generation). Serum PCB levels decreased significantly in the younger generation, from 179.1 (1.8) in the 1980 survey to 115.4 (2.0) in the 1995 survey (P<0.05). However, in the older generation, serum levels (ng/g lipid) did not change: 150.4 (1.6) in the 1980 survey and 180 (1.8) in the 1995 survey. Total PBDE serum levels (ng/g lipid) increased significantly during the 15 years, from 0.5 (3.5) to 1.8 (3.7) (P<0.05). At the Shimane site, PBDE serum levels (ng/g lipid) increased 20-fold, from 1.3 (4.8) to 26.0 (5.0). The serum levels of PCBs decreased in the younger generation but not in the older, although levels in daily intakes decreased significantly. Exposure levels of PBDEs appear to be increasing in an area-specific manner.

The endoplasmic reticulum stress response is stimulated through the continuous activation of transcription factors ATF6 and XBP1 in Ins2+/Akita pancreatic beta cells.

The dominant C96Y mutation of one of the two murine insulin genes, Ins2, causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet beta cell lines from heterozygous mice (Ins2+/Akita). Western blot analysis of endoplasmic reticulum (ER) molecular chaperones indicated that Grp78, Grp94 and Orp150 are significantly increased in Ins2+/Akita cells compared with wild-type (Ins2+/+) cells. Reporter gene assays using the human GRP78 promoter with or without the ER stress response element (ERSE) showed that Ins2+/Akita cells exhibit significantly stronger ERSE-dependent transcriptional activity than Ins2+/+ cells. Transient over-expression of the Ins2 C96Y mutant in wild-type beta cells induces a stronger ERSE-dependent stress response than does wild-type Ins2 over-expression. The ERSE-binding transcription factor ATF6 is strongly activated in Ins2+/Akita cells. The activity of a reporter containing the specific binding sequence of another ERSE-binding transcription factor, XBP1, is also enhanced in Ins2+/Akita cells. Levels of active forms of XBP1 mRNA and protein are both markedly elevated in Ins2+/Akita cells. These results indicate that this cell line is subject to continuous ER stress and that the Ins2 C96Y mutation induces the expression of ER chaperones through the activation of ATF6 and XBP1.