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Summary Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that recognizes a large number of xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), dioxins, and some endogenous ligands. Despite numerous investigations targeting AhR ligands, the precise physiological role of AhR remains unknown. Objective We explored novel AhR target genes, especially focused on inflammatory chemokine. Methods We treated (1) HaCaT, a human keratinocyte cell line, (2) normal human epidermal keratinocytes (NHEKs), and (3) mouse primary keratinocytes with AhR ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ; endogenous ligand) and benzo[a]pyrene (BaP; exogenous ligand). Then, we detected mRNA and protein of chemokine using quantitative RT-PCR and ELISA. We next clarified the relationship between AhR and chemokine expression using AhR siRNA. In addition, we measured serum chemokine levels in patients with Yusho disease (oil disease), who were accidentally exposed to dioxins in the past. Results We identified CC-chemokine ligand 5 (CCL5), a key mediator in the development of inflammatory responses, as the AhR target gene. AhR ligands (FICZ and BaP) significantly reduced CCL5 mRNA and protein expression in HaCaT cells. These effects were observed in NHEKs and mouse primary keratinocytes. AhR knockdown with siRNA restored CCL5 inhibition by AhR ligands. In addition, AhR ligands exhibited a dose-dependent suppression of CCL5 production induced by Th1-derived cytokines. Finally, serum levels of CCL5 in patients with Yusho disease, were significantly lower than in controls. Conclusion Our findings indicate that CCL5 is a target gene for AhR, and might be associated with the pathology of dioxin exposure.

水酸化ポリ塩化ビフェニル(OH-PCBs)は，人体内におけるPCBの主要代謝物である．LC/MS/MSを用いた血液試料中のOH-PCBs測定法の改良を行った．新しい粒径2µmのLCカラムを採用し，LC/MS/MS測定条件の最適化を行った．改良法では，5gの血液試料で従来法より感度が向上した．また，誘導体化のような試料の特別な処理も不要で，OH-PCB類，PCB類およびダイオキシン類の同一試料による同時定量も可能である．, Hydroxylated polychlorinated biphenyls (OH-PCBs) are well known as metabolites of PCBs in the human body. We improved a measurement method for OH-PCBs in blood samples using LC/MS/MS. A new 2µm particle column was used, and the analytical conditions for the LC/MS/MS measurements were optimized. The improved method is more sensitive than the conventional method for 5g blood samples. And, this method is effective at determining the concentrations of OH-PCBs, PCBs and dioxins from the same sample without special treatment of the sample such as derivatization.

Aim: Prenatal exposure to dioxins may result in many adverse health effects. However, the mechanisms by which dioxins are transferred from mother to fetus through the placenta are not well understood. The aim of this study was to investigate the differences in dioxin concentrations between maternal blood, the placenta, and cord blood in normal pregnant women, and to identify which individual congeners of these compounds are transferred from mother to fetus through the placenta. Material and Methods: Samples were collected from 19 pregnant Japanese women. Specific congeners of seven polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs), and four non-ortho polychlorinated biphenyls (PCBs) were analyzed. Results: The TEQ concentrations of PCDDs, PCDFs, and non-ortho PCBs were 8.03, 3.39, and 3.95 pg TEQ/g lipid, respectively, in the maternal blood; 8.78, 3.61, and 0.87 pg TEQ/g lipid in the placenta; and 4.33, 1.25, 1.08 pg TEQ/g lipid in the cord blood. Among specific congeners, 1,2,3,7,8-PentaCDD and 2,3,4,7,8-PentaCDF exhibited a placenta to maternal blood ratio greater than 1.0, while OctaCDD exhibited the greatest cord blood to placenta ratio. The cord blood to maternal blood ratio of total PCDDs was significantly higher than that of total PCDFs and total non-ortho PCBs. Conclusion: The dioxin concentration in cord blood was approximately half of the amount in maternal blood, despite congeners showing a high toxic equivalency factor accumulating in the placenta. PCDDs were transferred more readily than PCDFs and non-ortho PCBs from maternal blood to the fetus through the placenta.

Background: Studies on the association of maternal exposure to polychlorinated dibenzo-p-dioxin (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) with decreased birth weight in humans have produced conflicting results. In Japan in 1968, an accidental human exposure to rice oil contaminated with PCDDs, PCDFs, and PCBs, led to the development of Yusho disease. Objective: The Yusho cohort was used to evaluate the effect of maternal exposure to PCDDs, PCDFs, and PCBs on birth weight. Methods: Blood samples, obtained from 101 Yusho women (190 births) who gave birth after exposure, were analyzed for congeners of seven PCDDs, ten PCDFs, and four non-ortho PCBs. Results: Total PCDD TEQ (adjusted beta=−161.9 g; 95% CI, −265.3 to −58.6), total PCDF TEQ (adjusted beta=−105.9 g; 95% CI, −179.5 to −32.2), and total non-ortho PCBs (adjusted beta=−178.4 g; 95% CI, −318.3 to −38.5) levels were inversely associated with birth weight. Significant inverse associations with birth weight were also found for total PCDD TEQ, total PCDF TEQ, and total non-ortho PCB TEQ levels among male, but not female, infants. Significant inverse associations with birth weight were also found for nine congeners among all infants; the adjusted beta coefficients were largest for 1,2,3,6,7,8-HxCDD and smallest for 2,3,4,7,8-PeCDF. Conclusion: In the setting of exposure to high levels of dioxins, maternal blood levels of PCDDs, PCDFs and PCBs are associated with lower birth weight in Yusho patients. The association exhibited gender-specific differences, as male infants are more susceptible than females to growth restriction induced by in utero dioxin exposures. Keywords: Birth weight, Dioxin, Environmental exposure, Gender, Maternal blood, Yusho

In 1968, the contamination of cooking oil by heat-degraded polychlorinated biphenyls (PCBs) caused a case of mass poisoning, the so-called Yusho incident. The cause of Yusho disease is thought to be ingested toxic substances, including not only PCBs but polychlorinated dibenzofuran (PCDFs) in Kanemi rice oil. Extensive studies have been performed since 1995 by the Yusho study group involving follow-up surveys of human blood concentrations of the casual compounds in Yusho patients as well as clinical trials of the acceleration of the excretion of these compounds in Yusho patients. We have previously measured dioxin concentrations in the blood of 649 Yusho patients in annual medical examinations from 2001 to 2009. We determined that the concentrations of 2,3,4,7, 8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF, and 3,3',4,4',5,5'-HxCB (#169) in the blood of Yusho patients were more than twice as high levels to as those of normal controls. In this study, we compared the concentrations of 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7, 8-HxCDF, and 3,3',4,4',5,5'-HxCB (#169) in the blood of male Yusho patients with those of female Yusho patients. As a result, it was found that the mean concentrations of 2,3,4,7,8-PeCDF 1,2,3,4, 7,8-HxCDF 1,2,3,6,7,8-HxCDF, and 3,3',4,4',5,5'-HxCB (#169) in female Yusho patients were 2.9, 3.7, 2.4, and 1.3 times higher, respectively, than those of male Yusho patients.

We performed PCB congener specific analysis using HRGC/HRMS and determined their concentrations and blood chromatogram classifications in 275 individual blood samples collected during annual Yusho examinations between 2007 and 2010. When both blood total PCB concentrations and blood chromatogram classifications were compared among eleven Yusho patients undergoing examinations between 2007 and 2010, the longitudinal concentrations and classifications in the respective individuals hardly changed over these years. In a subset of persons suspected of Yusho, it was found that the mean total blood concentrations of three index congeners, that is, 2, 3', 4, 4', 5-pentaCB (PCB118), 2, 2', 4, 4', 5, 5'-hexaCB (PCB153), 2, 3, 3', 4, 4', 5-hexaCB (PCB156) and mean blood total PCB concentrations gradually decreased between 2007 and 2010. Mean concentration of PCB118 in blood from persons suspected of Yusho was calculated as 0.035 ng/g in the 2010 examination, which was 36% lower than the mean value of 0.054 ng/g in the 2007 examination: the concentration of PCB118 showed the highest degree of decrease among the three index congeners. Among persons suspected of Yusho, occupational rates of persons with blood PCB concentrations below one ppb in the entire population increased from approx. 50% in 2007 to approx. 70% in 2010. The lowest concentration of PCB156 in blood of persons suspected of Yusho in 2010 was 0.43 pg/g, which was almost equivalent to the limit of the determination value on HRGC/HRMS, S/N = 10.

In this study, we analyzed polychlorinated biphenyls (PCBs) in the blood of Yusho patients collected from medical check-ups, which were conducted in 2012. The results show that 65 PCB isomers, not including non-ortho PCBs, were detected in the blood samples, and the total concentration was 620 ng g(-1) lipid. This value was comparable to the concentration in blood samples collected in 2005, and indicated that PCB concentrations in Yusho patients remained unchanged from 2005 to 2012. Here, we focused on major and specific PCB isomers in the blood samples of Yusho patients and normal controls. Examples of the former include hexaCB-153, hexaCB-138, and heptaCB-180, which are detected in human blood, while the latter include hexaCB-156, hexaCB-157, and heptaCB-189, and are highly detected in the blood of Yusho patients. Additionally, we tried to determine why the specific isomers were highly accumulated in the blood of Yusho patients as compared to the normal controls. We therefore analyzed these isomers in the contaminated rice oil, and found that the concentrations of hexaCB-156, hexaCB-157, and heptaCB-189 were 1800, 450, and 190 ng g(-1), respectively. Notably, previous studies indicated that these isomers might not be easily metabolized in humans. Therefore, these findings demonstrated that these isomers were highly accumulated in the blood of Yusho patients.

We measured PCBs of blood of 154 people in the Yusho medical check-up from January 2011 through December 2014 and analyzed their PCB blood patterns. Eleven examinees were newly certified as Yusho patients during these 4 years. Of these 11, we identified three Yusho patients with the A pattern, five with the B pattern, and three with the BC pattern. We found no Yusho patients with the C pattern. The B pattern was found in 27 of the 154 examinees, such that the probability that an examinee with the B pattern would be certified as a Yusho patient was about 20%. Since 2012, any family members living with a Yusho patient have also been certified as Yusho patients. The number of such family cohabitants was 16, among whom the PCB pattern was uniformly the C pattern. There were 20 examinees born after 1989, and all those examinees were Yusho-suspected persons. The total PCB concentrations of this younger-age group was less than 0.5 ng/g on average and was low compared with the normal controls.

We compared concentrations of PCB congeners between conventional 80 degrees C alkali digestion method and 30 degrees C alkali digestion method using 22 individual blood samples collected during 2009 annual Yusho examinations. The mean concentration of tri-CB in 30 degrees C alkali digestion method was lower than that of conventional method. However, concentration of total PCBs, 2,3',4,4',5-pentaCB (PCB118), 2,2',4,4',5,5'-hexaCB (PCB153) and 2,3,3',4,4', 5-hexaCB (PCB156), which were used as index for Yusho diagnosis, were equivalent between the methods. It was considered that the 30 degrees C alkali digestion method was available for blood PCB congener specific analysis in Yusho diagnosis.

We developed an analytical method for accurately determining the concentrations of dioxin and polychlorinated biphenyls(PCBs) in a blood volume of 5 g. Inter-laboratory crosscheck is available for maintaining the quality of dioxin and PCB analysis. We carried out the crosschecks of dioxin and PCB analysis in the blood with domestic analysis organizations. Two crosschecks each of dioxin and PCB analysis were carried out from 2007 through 2011. As a result, the findings of our analysis accorded well with the analyses of the other organizations. We believe that our analytical method is sufficiently reliable.

Prenatal exposure to dioxins may result in many adverse health effects. However, the mechanisms by which dioxins are transferred from mother to fetus through the placenta are not well understood. The aim of this study was to investigate the differences in dioxin concentrations between maternal blood, the placenta, and cord blood in normal pregnant women, and to identify which individual congeners of these compounds are transferred from mother to fetus through the placenta.Samples were collected from 19 pregnant Japanese women. Specific congeners of seven polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs), and four non-ortho polychlorinated biphenyls (PCBs) were analyzed.The TEQ concentrations of PCDDs, PCDFs, and non-ortho PCBs were 8.03, 3.39, and 3.95 pg TEQ/g lipid, respectively, in the maternal blood; 8.78, 3.61, and 0.87 pg TEQ/g lipid in the placenta; and 4.33, 1.25, 1.08 pg TEQ/g lipid in the cord blood. Among specific congeners, 1,2,3,7,8-PentaCDD and 2,3,4,7,8-PentaCDF exhibited a placenta to maternal blood ratio greater than 1.0, while OctaCDD exhibited the greatest cord blood to placenta ratio. The cord blood to maternal blood ratio of total PCDDs was significantly higher than that of total PCDFs and total non-ortho PCBs.The dioxin concentration in cord blood was approximately half of the amount in maternal blood, despite congeners showing a high toxic equivalency factor accumulating in the placenta. PCDDs were transferred more readily than PCDFs and non-ortho PCBs from maternal blood to the fetus through the placenta.

Maternal exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) may result in adverse health effects in their children. In Japan in 1968, an accidental human exposure to rice oil contaminated with PCDDs, PCDFs, and PCBs, led to the development of Yusho disease. Yusho mothers delivered descendants with low birth weights and hyperpigmented skin and mucosa, which are characteristic of fetal Yusho disease (FYD). The Yusho cohort was used to evaluate the effect of maternal exposure to PCDDs, PCDFs, and PCBs on the development of FYD. Blood samples, obtained from 64 Yusho mothers (117 descendants: 10 with FYD and 107 without FYD), were analyzed for congeners of seven PCDDs, 10 PCDFs, and four coplanar PCBs. We investigated the association between the maternal estimated blood levels of dioxins at delivery and the risk of fetal Yusho disease. We also studied the differences in dioxin blood levels in 24 mother–descendant pairs (5 with FYD and 19 without FYD). The estimated levels of total PCDD TEQ, total PCDF TEQ, total coplanar PCB TEQ, and total TEQ in the maternal blood at delivery were associated with significantly increased risk of FYD. The odds ratios, which present the risk of FYD for a 10-fold increase in blood dioxin, were largest for 1,2,3,6,7,8-HexaCDD (odds ratio = 28.6, 95% confidence interval = 1.67–489.9, p = 0.02). The levels of 1,2,3,6,7,8-HexaCDD in both the Yusho mothers and their descendants with FYD were higher than the levels in those without FYD. These findings suggest that 1,2,3,6,7,8-HexaCDD is the most important causative congener for the development of FYD.

The polychlorinated quaterphenyl (PCQ) concentrations in blood are important discriminative parameters in yusho patient. In this study, a rapid analytical method for PCQ using different diameter capillary column (rapid-Rtx65TG) with high-resolution gas chromatograph high-resolution mass spectrometer (HRGC/HRMS) instead of the gas chromatograph electron capture detector (ECD/GC) was developed. Using different diameter capillary columns, the analysis time of the HRGC/HRMS was drastically shortened, and the detection sensitivity was improved. In the rapid-Rtx65TG column, a small-bore capillary column (length 1m, I.D. 0.1mm) was connected with the inlet side of the GC, and behind that column, a large-bore capillary column (length 15mm, I.D. 0.53mm) for octadecachloroquaterphenyl (ODCQ) analysis was connected. In the HRGC/HRMS measurement of ODCQ by the rapid-Rtx65TG column, the minimum limit of detection for the apparatus was 0.4 pg, and the minimum limit of determination for the blood was 0.008 ppb. On ECD/GC in the conventional method and HRGC/HRMS in this study, the PCQ concentration in blood including yusho patients and yusho suspected persons was almost equivalent.

Objectives Little information is available on conditional survival among Yusho patients, who were accidentally exposed to PCBs and other dioxin-related compounds. In this study, we estimated relative survival among Yusho patients to quantify time trends in excess mortality compared to the general population. Methods A total of 1664 Yusho patients (860 males, and 804 females) were analyzed as Yusho cohort subjects. Relative survival ratio (RSR) was calculated as a measure of patient survival. Results Overall, 1-, 5-, 10-, and 15-year RSRs were 1.00 (95% confidence interval (CI): 0.99, 1.00), 1.00 (95% CI: 0.99, 1.01), 0.99 (95% CI: 0.98, 1.00), and 0.99 (95% CI: 0.98, 1.01), respectively. We did not observe meaningful increases or decreases in RSRs in either sex, which remained the same in all age groups for 1-, 5-, 10-, and 15-year RSRs. Conclusions This study provides quantitative evidence that Yusho patients have no significant difference in relative survival compared with the general Japanese population. Our results suggest that PCBs and dioxin exposure confers no excess mortality. This information may be important for both the clinical management of and patient coping with Yusho disease.

Accumulated maternal dioxins are passed onto the fetus and neonate via the placenta and maternal milk. In Japan in 1968, an accidental human exposure to rice oil contaminated with polychlorinated biphenyls (PCBs) and other dioxin-related compounds, such as polychlorinated dibenzofurans (PCDFs), led to development of Yusho oil disease. We investigated differences in blood dioxin concentrations in mother-children pairs affected by the Yusho incident. From 2002 to 2008, blood samples were collected from 26 pairs of Yusho mothers and their children (19 mothers, 26 children). Specific congeners of seven polychlorinated dibenzo-p-dioxins (PCDDs), ten PCDFs, and four non-ortho PCBs were analyzed. The children had significantly lower TEQ concentrations of PCDDs, PCDFs, and coplanar PCBs compared to their mothers. The mother-child difference in blood concentrations varied with the congeners; the largest for 2,3,4,7,8-pentaCDF and the smallest for 1,2,3,4,6,7,8-heptaCDD. The level for 2,3,4,7,8-pentaCDF, which characterizes Yusho oil disease, was approximately 17-30 times higher in the mothers than in the general population, whereas there were no significant differences between children in the formula-fed group and the general population. In contrast, the mean level for 2,3,4,7,8-pentaCDF in the breast-fed group was approximately 1.5 times, (range 0.5-6.5 times) higher than that in the general population. Over 30 years after the Yusho incident, the mean blood dioxin levels in the offspring were only a fraction of the levels in their mothers. This is more consistent with exposure via breast milk than via transplacental transfer in the Yusho incident.