This study attempts to relate the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent (TEQ) level with certain responses including the catalytic activities and expression of hepatic cytochrome P450 (CYP) 1A and CYP1B in wild population of Baikal seal (Pusa sibirica). We isolated full-length CYP1A1, 1A2, and 1B1 cDNAs, which encode proteins of 516, 512, and 543 amino acids, respectively. Immunochemical analysis demonstrated that a cross-reactive protein with polyclonal antibody against rat CYP1A1 or CYP1B1 was detected in the seal liver. Total TEQ levels showed significant positive correlations with expression levels of CYP1A1, 1A2, and 1B1 mRNAs, and further with both CYP1A- and CYP1B-like proteins, indicating chronic induction of these CYP isozymes by TEQs. The 50% effective concentration for CYP1A-like protein induction was estimated to be 65 pg TEQ/g wet weight. To evaluate the potential of congener-specific metabolism, profiles of negative correlations between the concentrations of eachcongener normalized to a relatively recalcitrant congener, PCB169, and CYP1A-like protein levels were also estimated. Significant negative correlations of 2,3,7,8-tetrachlorodibenzofuran and PCB77 to CYP1A-like protein expression may possibly be due to the preferential metabolism of these congeners. Anti-rat CYP1A1 and CYP1B1 antisera equivalently inhibited ethoxyresorufin O-deethylase (EROD) activity in the seal microsomes, suggesting that both CYPs are involved in EROD activity. Hepatic EROD revealed an increasing trend at lower TEQs, but a declining trend at higher levels, implying a catalytic inhibition of CYP1A and CYP1B. Furthermore, ratios of CYP1B1/CYP1A1 mRNA expression levels increased with TEQs, indicating the enhanced risk of carcinogenicity by preferential induction of CYP1B1 by TEQs in the liver.

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We have previously reported that high accumulation of dioxins and related compounds induced cytochrome P450 (CYP 1s) isozymes in the liver of wild Baikal seals, implying the enhanced hydroxylation of polychlorinated biphenyls (PCBs). The present study attempted to elucidate the residue concentrations and patterns of PCBs and hydroxylated PCBs (OH-PCBs) in the livers of Baikal seals. The hepatic residue concentrations were used to assess the potential effects of PCBs and OH-PCBs in combination with the analyses of serum thyroid hormones, hepatic mRNA levels, and biochemical markers. The hepatic expression levels of CYP1 genes were positively correlated with the concentration of each OH-PCB congener. This suggests chronic induction of these CYP1 isozymes by exposure to PCBs and hydroxylation of PCBs induced by CYP 1s. Hepatic mRNA expression monitoring using a custom microarray showed that chronic exposure to PCBs and their metabolites alters the gene expression levels related to oxidative stress, iron ion homeostasis, and inflammatory responses. In addition, the concentrations of OH-PCBs were negatively correlated with L-thyroxine (T4) levels and the ratios of 3,3',5-triiodo-L-thyronine (T3)/reverse 3,3',5'-triiodo-L-thyroninee (rT3). These observations imply that Baikal seals contaminated with high levels of OH-PCBs may undergo the disruption of mechanisms related to the formation (or metabolism) of T3 and T4 in the liver.

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In what has become known as the Yusho incident, thousands of people in western Japan were poisoned by the accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds. In this study, we investigated the accumulation patterns of 69 PCB congeners in the blood of Yusho patients in comparison with those of non-exposed controls. The blood samples were collected at medical check-ups in 2004 and 2005. To compare the patterns of PCB congeners, we calculated the concentration ratio of each congener relative to the 2,2',4,4',5,5'-hexaCB (CB153) concentration. The concentration ratios of tetra- and penta-chlorinated congeners in the blood of Yusho patients were significantly lower than those of controls. To examine the cytochrome P450 (CYP)-dependent metabolic potential of the 2,3',4,4'5-pentaCB (CB118), CB153, and 2,3,3',4,4'5-hexaCB (CB156) congeners, we conducted PCB-CYP (CYP1A1, CYP1A2, CYP2A6, and CYP2B6) docking simulation by in silico analysis. The docking models showed that human CYP1A1, CYP2A6, and CYP2B6 isozymes have the potential to metabolize CB118 and CB153. On the other hand, it was inferred that CB156 is difficult to be metabolized by these four CYP isozymes. These results indicate that CYP1 and CYP2 isozymes may be involved in the characteristic accumulation patterns of PCB congeners in the blood of Yusho patients.

Summar y Since 2009, Ministry of the Environment, Japan has discussed the issues in using "Whole Effluent Toxicity (WET) test" from both technical and legal standpoints. One of the issues is the current expensive cost of the WET test. Therefore, it is necessary to develop a simpler and cheaper toxicity test. In this study, we developed a small-scale algal growth inhibition test (small-scale method) to test for toxicity. Among several test vessel sizes and test medium volumes examined for the small-scale method, the growth rate using a 50 mL glass Erlenmeyer flask with 20 mL of test medium was found to be the most comparable to that of the existing draft protocol of the algal growth inhibition test (the existing method). The developed small-scale method was first evaluated by testing of potassium dichromate and 3, 5-dichlorophenol. In the testing of potassium dichromate, it was shown that the IC25 and control coefficient of variation (CV) of the growth rate were at acceptable levels compared with findings from the Japanese laboratory. Furthermore, the no observed effect concentration (NOEC), IC25, and IC50 of the wastewater samples by the small-scale method were almost the same as those by the existing method, although the detection power of the small-scale method was somewhat lower. We investigated 25 effluent samples by the developed small-scale method. Algal growth inhibition was confirmed with 8 wastewater samples in the lowest concentration group (20% concentration group). Our results suggest that the developed small scale method is effective for identifying the tendency of algae growth inhibition. In addition, the developed small-scale method was able to test multiple samples simultaneously. Therefore, it is possible that Toxicity Identification Evaluations (TIEs) can be performed efficiently by our new developed small-scale method.

Summar y In this study, we examined a fish survey method using environmental DNA (eDNA) metabarcoding in a river, and the relationship between the fish survey results and water quality detected in eDNA was analyzed. eDNA surveys and fishing surveys were conducted in summer and winter, targeting three sites on three rivers in Fukuoka prefecture. Our comparison of riffles and pools and our examination of the appropriate places to collect eDNA samples revealed that the detected fish species were not completely consistent between the two kinds of surveys and there was no ecological features common to detected fish species. Therefore, sampling at multiple locations is expected to reduce detectable fish dropouts. More fish species were detected in the eDNA survey than in the collection survey at all times. However, the results also suggested that the DNA of fish species living upstream may remain at sampling site, and benthic fish species tend to be difficult to detect by eDNA surveys. In addition, the influence of water quality on the characteristics of the survey site were analyzed, since marine species were detected in some river eDNA surveys. The results suggested that domestic drainage is likely to affect the characteristics in winter, when the amount of water is small, and the change in concentration of linear alkylbenzene sulfonic acid and its salts and the change of the ratio of chloride ions may be used as an index of domestic drainage.

Compound-specific isotope analysis (CSIA) can be used to examine the source and transformation processes of organic pollutants in the environment. We performed a carbon stable isotope analysis of polychlorinated biphenyl (PCB) congeners in the PCB heat-transfer medium (heat medium) and the original Kanechlor-400 (KC-400, a commercial brand of PCBs) involved in the Yusho incident. The main purpose is to investigate whether isotope fractionation occurred in the rice oil deodorization process that caused the incident. The carbon isotope ratios (δC values) of the targeted PCB congeners ranged from - 29.39 to - 27.00‰ in the heat medium and from - 28.77 to - 27.05‰ in the original KC-400. No significant differences were found in the δC values, suggesting carbon isotope fractionation did not occur for the targeted PCB congeners in the heat medium when deodorization of rice oil occurred at temperatures above 200 °C. Furthermore, we also conducted a congener-specific analysis of 64 PCB congeners found in the heat medium and rice oil contaminated by it. The total PCB congener concentrations were 503 mg/g in the heat medium and 81 μg/g in the rice oil. The concentrations of the highly chlorinated congeners were significantly lower in the heat medium than in the original KC-400, and the compositional ratios of the lowly chlorinated congeners were relatively lower in the rice oil than in the heat medium. These results suggest that the PCB congener patterns gradually changed from that of the original KC-400 in the deodorization process and subsequent contamination into the rice oil. Thus, a combination of CSIA and congener-specific analysis is a new approach for investigating the changing PCB congener profiles in samples from the Yusho incident.