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Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the Bam HI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10 −8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy ( n = 10) and those who did not ( n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10 −8 per base pair while the latter had 9.0 ± 4.5 × 10 −8 per base pair ( P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Aberrant DNA methylation, induced by aging and accelerated by chronic inflammation, accumulates in normal tissue. Accumulation of DNA methylation in multiple genes, namely epimutation burden, is closely associated with cancer risk. Indeed, we demonstrated that the measurement of the methylation level of a marker gene in normal tissue is useful for cancer risk prediction of metachronous cancers, by a multicenter prospective study with gastric cancer patients (Asada, Gut, 64:388, 2015: Maeda, Gut, 66:1721, 2017). In this study, we attempted to predict the risk of primary gastric cancer by performing a new multicenter prospective study. We recruited 1,880 healthy people, after Helicobacter pylori eradication, with a clinically high risk of gastric cancer. Biopsy specimens were obtained from the antrum and body, and the methylation level of RIMS1, which is a preselected epigenomic risk marker for this study, was measured in 1,757 participants. The participants are annually being followed-up to detect primary gastric cancer, for 5-8 years. Currently, the median follow-up is 4.05 years, and 27 people have developed primary gastric cancers (as of September 2022). The results of statistical analysis are being determined. This study will establish the clinical utility of a cancer risk diagnosis by epimutation burden in normal tissue, and is promising for application in various inflammation-associated cancers. Citation Format: Harumi Yamada, Masahiro Maeda, Seiichiro Abe, Taichi Shimazu, Takayuki Ando, Kazunari Murakami, Shinji Tanaka, Takao Maekita, Osamu Inatomi, Ken Sugimoto, Tetsuro Kawagoe, Nobutake Yamamichi, Toshikazu Ushijima. Cancer risk diagnosis by epimutation burden: a multicenter prospective study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6014.

Introduction Aims of this study were (a) to assess the development ratio of hyponatremia during treatment with linezolid and (b) to evaluate the relationship between the risk of hyponatremia and linezolid exposure and patient background. Method Clinical data including linezolid serum concentrations and serum sodium values were collected at Toyama University Hospital and Kyorin University Hospital. Data from 89 patients were used for the analysis, and a nadir serum sodium level ≤130 mmol/L during the treatment with linezolid was defined as hyponatremia. Mann-Whitney's U test was used to evaluate the effects of the area under the time-concentration curve (AUC) of linezolid at the nadir sodium level, clinical characteristics (e.g. laboratory data), and baseline serum sodium levels on the development of hyponatremia. Results The hyponatremia was occurred in 21 of 89 patients (23.6%). Data are compared for baseline and nadir serum sodium levels of patients with and without hyponatremia. In both groups, nadir serum sodium levels were significantly different from those of the baseline values (P Conclusions Linezolid exposure, age, and baseline sodium levels were detected as the risk factors for linezolid-related hyponatremia. Our findings suggest that regular monitoring of serum sodium levels is desirable during treatment with linezolid, especially for the elderly and patients with low serum sodium levels before the start of linezolid administration.

Background and aim The reliable method to stratify the gastric cancer risk after Helicobacter pylori eradication remains an elusive goal. Methods Mass eradication of H. pylori began in 2004 in a high-risk population. After eradication, a screening program involving first-stage serological tests (pepsinogen-I, pepsinogen-II, H. pylori immunoglobin G, and gastrin-17) and second-stage endoscopic examination was launched in 2015-2018. Index lesions included gastric cancer or extensive premalignant lesions. We evaluated the performance of the serological tests to "rule in" and "rule out" the risk based on positive and negative likelihood ratios, respectively. The methylation levels of microRNA-124a-3 in the stomach were measured to indicate genetic damage. Results Among 6512 invited subjects, 3895 (59.6%) participated. Both gastrin-17 and pepsinogen tests were normal in 3560 (91.4%) subjects; 206 (5.3%) gastrin-17 and 129 (3.3%) pepsinogen tests were abnormal. Years after eradication, the severity of gastritis had fallen greatly, and extensive premalignant lesions or gastric cancer frequently occurred in newly non-atrophic-appearing mucosa. Pepsinogen testing could moderately predict atrophic gastritis (positive likelihood ratio: 4.11 [95% confidence interval: 2.92-5.77]; negative likelihood ratio: 0.14 [0.10-0.19]). Gastrin-17 was not useful (0.66 and 1.20, respectively). However, pepsinogen testing poorly predicted the index lesions (2.04 [1.21-3.42] and 0.57 [0.34-0.95]). DNA methylation levels in the post-eradication mucosa were more discriminative for predicting index lesions (3.89 [2.32-6.54] and 0.25 [0.15-0.42]). Conclusions After eradication, pepsinogen false-negative results become more frequent because histology is improved but genetic damage may persist. Direct testing for genetic damage offers better discrimination.

Direct-acting antivirals, asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are known to be mainly metabolized by CYP3A enzymes; however, the differences in the detailed metabolic activities of CYP3A4 and CYP3A5 on these drugs are not well clarified. The aim of the present study was to elucidate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of ASV, DCV, and BCV, as well as the effect of CYP3A5*3 genetic variant in vitro. The amount of each drug and their major metabolites were determined using LC-MS/MS. Recombinant CYP3As and CYP3A5*3-genotyped human liver microsomes (CYP3A5 expressers or non-expressers) were used for the determination of their metabolic activities. The contribution of CYP3A5 to ASV metabolism was considerable compared to that of CYP3A4. Consistently, ASV metabolic activity in CYP3A5 expressers was higher than those in CYP3A5 non-expresser. Moreover, CYP3A5 expression level was significantly correlated with ASV metabolism. In contrast, these observations were not found in DCV and BCV metabolism. To our knowledge, this is the first study to directly demonstrate the effect of CYP3A5*3 genetic variants on the metabolism of ASV. The findings of the present study may provide basic information on ASV, DCV, and BCV metabolisms.

Purpose The cytochrome P450 (CYP) 3A family of enzymes metabolize the majority of clinically used drugs. CYP3A4 and CYP3A5 are the two major CYP3A isoforms, but exhinbit different substrate specificity. The aim of this study was to establish a simple screening method to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro. Methods A screening method was developed based on competitive inhibition using luciferin-PPXE (L-PPXE), a luminogenic CYP3A substrate. CYP3cide, tacrolimus, and midazolam were selected as standard compounds metabolized by CYP3A4 or CYP3A5. Nine clinically-used drugs were evaluated for their abilities to inhibit luminescence resulting from L-PPXE metabolism. Appropriate reaction conditions for the screening method were determined using recombinant CYP3A4 and CYP3A5. Results A significant decrease in luminescence resulting from L-PPXE metabolism by CYP3A4 and CYP3A5 was observed only for drugs reported to be metabolized by CYP3As. The substrate specificities of CYP3A4 or CYP3A5 for the proposed CYP3A substrates using our screening method were consistent with those of previous reports or available drug information from pharmaceutical companies. The reaction volume for this method was 50 μL, and the time required for the entire procedure was 70 min. Furthermore, this screening can be performed using a single tube with minimal training. Conclusions Through the establishment of our screening method in the present study, we are sure it is useful to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro.

The CpG island methylator phenotype (CIMP) is associated with prognosis and drug sensitivity in multiple cancer types. In gastric cancer, the CIMP is closely associated with Epstein-Barr virus (EBV) infection and AT-rich interactive domain 1A (ARID1A) mutations, a component of the SWI/SNF chromatin remodeling complex. However, the involvement of SWI/SNF defects in CIMP induction has been unclear. In this study, we demonstrate a causal role of ARID1A loss-of-function in CIMP induction. Mutations of SWI/SNF components, especially ARID1A, was associated with the CIMP, as well as EBV infection, in gastric cancers, and also in uterine endometrial and colorectal cancers, which are not affected by EBV infection. Genome-wide DNA methylation analysis showed that ARID1A knockout (KO) in cultured 293FT cells and gastric epithelial cells, GES1, induced aberrant DNA methylation of a substantial number of CpG sites. DNA methylation was induced at genomic regions with high levels of pre-existing histone H3 lysine 27 trimethylation (H3K27me3) and those with acquired H3K27me3 by ARID1A KO. These results showed that the ARID1A mutation induced aberrant DNA methylation, and this is likely to be one of the potential mechanisms of CIMP induction.

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.

Aberrant DNA methylation can cause silencing of tumor-suppressor genes and can be causally involved in cancer development and progression. Aberrant DNA methylation can be detected using only DNA, which enables cancer detection in various specimens. DNA methylation can predict that a gene will never be expressed, even if its expression is induced by drug treatment. Methylation level of a specific gene may be associated with drug response or patient prognosis. Aberrant methylation in normal tissues can be utilized for cancer risk diagnosis. Therapeutically, aberrant methylation can be removed using demethylating drugs such as 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine). These drugs are used at a relatively low dose with strong biological effect for a prolonged period, and they induce reprogramming of a diseased epigenome. They are currently used in practice for hematological malignancies and in clinical trials for solid tumors. The use of an appropriate dose and schedule is a key to success.

Linezolid (LZD) is an antimicrobial that is commonly used for treatment of vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus infections. However, the development of thrombocytopenia, one of the most frequent adverse side effects of this antimicrobial, can lead to discontinuation of LZD treatment. While clinical studies indicate that risk factors for the development of LZD-induced thrombocytopenia include treatment for >14 consecutive days, renal dysfunction, and chronic liver disease, the fundamental mechanism governing the pathogenesis of this disorder remains unclear. In this study, we aimed to elucidate the mechanism of LZD-induced thrombocytopenia by investigating the impact of LZD treatment on platelet destruction and production using rat platelet-rich plasma (PRP) and human immortalized cell lines, respectively. Compared to the control population, an increase in lactate dehydrogenase release was not detected upon exposure of rat PRP to varying concentrations of LZD, indicating that this compound is not cytotoxic towards platelets. Meanwhile, LZD treatment resulted in a significant dose-dependent increase in the proliferation of HEL human erythroleukemia and MEG-01 human megakaryoblast cells in vitro, but did not influence the differentiation of these cell lines. Lastly, LZD treatment yielded elevated levels of phosphorylation of myosin light chain 2 (MLC2), which regulates platelet release, in MEG-01 cells. Based on these results, we speculate that LZD induces thrombocytopenia by promoting MLC2 phosphorylation and thereby suppressing the release of platelets from mature megakaryocytes. These findings provide the first insight into the mechanism of LZD-mediated thrombocytopenia and may facilitate the development of strategies to treat and/or prevent this disease.

Risk prediction for gastric cancer (GC) is important, especially for H. pylori-eradicated individuals whose number is rapidly increasing in Japan. For accurate cancer risk prediction, analysis of epigenetic changes, particularly aberrant DNA methylations, has a great potential. It is induced in the gastric mucosa by H. pylori infection, persists for life, and is causally involved in gastric carcinogenesis. The DNA methylation levels in individuals without current H. pylori infection correlate with GC risk and have a greater impact than that of accumulated point mutations. A methylation marker is necessary to assess the overall epigenomic damage accumulated in the genome of gastric epithelial cells. Initially, CpG islands methylated in GC cells were used. More informative markers were then isolated by an analysis of the gastric mucosa of gastric cancer patients and healthy individuals. Finally, highly informative markers unaffected by contaminating blood cells have been developed using an advanced technology and a screening algorithm. With an aim of bringing epigenetic cancer risk diagnosis into practice, we first conducted a multicenter prospective cohort study for risk prediction of metachronous GC among GC patients who had undergone endoscopic treatment and achieved the first proof of concept. We are currently conducting a new, nationwide study for risk prediction of primary GC among healthy H. pylori-eradicated individuals. Epigenetic cancer risk diagnosis, which was initially developed for GC and potentially applicable to other inflammation-associated cancers, has a great potential to contribute to precision medicine.

Estrone-3-sulfate (E1S) is thought to be a major estrogen precursor in estrogen receptor (ER)-positive breast cancer. Since E1S is a hydrophilic compound, the uptake of E1S into cancer cells is probably mediated by transporters, such as organic anion-transporting polypeptide (OATP, SLCO) family. In this study, we investigated the relationship between expression of OATP2B1 and cell proliferation in ER-positive breast cancer. Cell-based assays were carried out in MCF-7 cells both with and without overexpression of OATP2B1. Normal breast and tumor tissues were collected and used in this study. Cell proliferation, ER-mediated transcriptional activities and estradiol secretion were stimulated by addition of E1S to the culture medium of MCF-7 cells. These stimulatory effects were significantly greater in MCF-7 cells overexpressing OATP2B1 than in control cells. The expression level of SLCO2B1 mRNA was significantly correlated with histological grade, Ki-67 labelling index and mRNA expression of steroid sulfatase. The expression level of SLCO2B1 mRNA in luminal B-like cancers was higher than that in luminal A-like cancers. Uptake of E1S resulted in down-regulation of ERα protein and induction of Ki-67 in MCF-7 cells. The present study suggests that OATP2B1 is involved in cell proliferation by increasing the amount of estrogen in ER-positive breast cancer cells.

Although the number of marine protected areas (MPAs) for stock management has increased, movements or differences in population structure of a target species between an MPA and surrounding fishing areas have rarely been considered in stock biomass estimations. We developed a surplus production model considering seasonal movements between two areas; the model was applied to Sea Ravens Hemitripterus villosus off Fukushima, where almost all fishing has been prohibited since the 2011 accident at the Fukushima Dai-ichi Nuclear Power Plant. We predicted future biomass by using CPUE data from coastal gill-net fishing and offshore bottom trawl fishing in 2000 to 2009. The model reflected the seasonal coastal–offshore movements of Sea Ravens well, and it predicted increasing Sea Raven biomass in both areas, which was validated by the CPUEs observed after 2010—including those for trial bottom trawl fishing that occurred within limited offshore areas after the accident. Our results indicate that the newly de...

Clenbuterol is a long-acting β2-adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (-)-R-enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC-MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1 ml volumes of plasma were precisely quantified at concentrations as low as 0.25 ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2 mg/kg) in rats were significantly different. The distribution volume of (-)-R-clenbuterol (9.17 l/kg) was significantly higher than that of (+)-S-clenbuterol (4.14 l/kg). The total body clearance of (-)-R-clenbuterol (13.5 ml/min/kg) was significantly higher than that of the (+)-S-enantiomer (11.5 ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (-)-R- and (+)-S-enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (-)-R-clenbuterol was higher than that of the (+)-S-enantiomer. The fractions of free (non-protein-bound) (-)-R- and (+)-S-clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding.

Changes in the expression level and activity of cytochrome P450 (CYP) in the liver are caused by various factors and affect the pharmacokinetics of drugs. The purpose of this study was to determine whether the expression of CYP3A is affected by a high-fat diet. In addition, we examined whether the type of diet given to mice could produce changes in the expression level and activity of CYP3A. Mice were fed a purified diet containing 10 kcal% lard (control group) or 60 kcal% lard (HF group) or regular mouse chow containing 13 kcal% of fat (MF group) for 4 weeks. No significant differences were observed in the hepatic CYP3A protein expression level between the HF group and the control group. The CYP3A protein expression in the MF group was significantly higher than that observed in the control group. In the MF group, the area under the curve (AUC) of intraperitoneally administered triazolam was lower. Because lithocholic acid (LCA) is known to increase hepatic CYP3A expression, the levels of Clostridium sordellii and LCA in the feces were measured. In the MF group, the levels of Clostridium sordellii and LCA were higher. It has been demonstrated that a high-fat diet does not cause any changes in hepatic CYP3A expression. In addition, the different diets caused alterations in the enteric environment, which triggered changes in CYP3A expression. Therefore, it is necessary to carefully consider the type of feed while performing animal experiments to evaluate the pharmacokinetics of drugs.

Recent studies have reported that a high-fat diet during pregnancy exerts various effects on the foetus and newborn. The purpose of this study was to clarify the effects of a high-fat diet during pregnancy on the activity of hepatic cytochrome P450 (Cyp) 3a in offspring in mice. The protein expression level and activity of Cyp3a in the livers of 6-week-old mice born to mothers that were given a high-fat diet during pregnancy (HF group) decreased significantly compared with the Control group. Triazolam, which is a substrate of Cyp3a, was intraperitoneally administered to the mice in the HF group. Compared with the Control group, an increase in the area under the plasma concentration–time curve and a decrease in total clearance were observed in the HF group. The hepatic constitutive androstane receptor (CAR) mRNA expression level in the HF group was significantly lower than that in the Control group. An increase in phosphorylation of extracellular signal-regulated kinase (ERK) was also observed in the HF group. The results of this study revealed that a high-fat diet during pregnancy causes an increase in ERK phosphorylation and a decrease in the expression level of CAR in the livers of offspring, which leads to decreased Cyp3a expression and activity. The results suggest that individual differences in pharmacokinetics may not only be expressed by genetic predisposition but also by a mother’s living environment during pregnancy.

Chronic inflammation is deeply involved in the development of human cancers by inducing epigenetic alterations, such as aberrant DNA methylation. Among various inflammatory cytokines, the expression of Il1b, Tnf, and Nos2, is associated with induction of aberrant DNA methylation. However, the molecular mechanisms by which these cytokines induce aberrant DNA methylation remain unclear. Here, we show that the activation of the NF-kB signaling pathway, downstream of IL-1β and TNF-α, caused up-regulation of specific miRNAs, such as miR-20a, miR-26b, and miR-29c, and that these miRNAs caused repression of the Tet methylcytosine dioxygenases (Tet) genes, Tet1, Tet2, and Tet3. TET repression by overexpression of one of these miRNAs in cultured cells appeared to be insufficient for induction of aberrant DNA methylation as detected by an Infinium MethylationEPIC BeadChip array. However, triple knockout of TET genes induced strong DNA methylation at thousands of genomic loci. At the same time, exposure to nitric oxide, produced by Nos2, enhanced enzymatic activity of DNA methyltransferases (DNMTs). Treatment of cultured cells with nitric oxide induced weak DNA methylation at hundreds of genomic loci. These results show that both the repression of TET genes and enhancement of DNMT activity, induced by chronic inflammation, are critical for induction of aberrant DNA methylation. ''Vicious'' combination of such dysregulations might have a synergistic effect on induction of aberrant DNA methylation. Citation Format: Hideyuki Takeshima, Tohru Niwa, Harumi Yamada, Satoshi Yamashita, Mika Wakabayashi, Toshikazu Ushijima. Repression of TET genes and enhancement of DNA methyltransferase activity are critical for induction of aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5331.

Aberrant DNA methylation accumulated in normal tissues, namely methylation burden, is closely associated with risk of carcinogenesis, especially in cancers related to chronic inflammation. Methylation burden is known to be influenced by multiple factors, such as genetic factors and strengths of carcinogens. However, the impact of the duration of exposure to a carcinogen is still unclear. Here, using a Mongolian gerbil model of Helicobacter pylori (H. pylori)-induced chronic gastritis, we aimed to clarify the impact of the duration of exposure on the methylation burden in normal gastric tissues (gastric mucosae). Following infection with H. pylori, DNA methylation levels at four CpG islands, HE6, SA9, SB5, and SD2, increased, depending upon the exposure duration. After eradication of H. pylori, DNA methylation levels decreased, but tended to be higher in gastric mucosae with a longer infection period. DNA molecules with dense DNA methylation, but not those with sparse DNA methylation, increased depending upon the infection period. DNA methylation levels at one of the four CpG islands, SA9, tended to be higher in the gastric mucosae of gerbils infected with H. pylori than in those of non-infected gerbils, even 50 weeks after eradication. These results showed, for the first time, that the methylation burden in normal tissues is influenced by the duration of exposure to a carcinogenic factor. Citation Format: Harumi Yamada, Hideyuki Takeshima, Tohru Niwa, Takeshi Toyoda, Satoshi Yamashita, Toshikazu Ushijima. The methylation burden is determined by the duration of exposure to carcinogenic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5340.

It is clarified that the ozone consumption during the initial stage of ozonation before dissolved ozone appears is the same value as that during the initial 20 seconds in batch mode experiment. This initial ozone consumption was defined as initial ozone demand (IOD) in this study. IOD of secondary effluent was shown to be 0.3–0.5 mgO3/mgC0. More than 90% of 17β-estradiol (E2) was reduced, and formation of ozonation by-products was controlled within this ozone demand. This state can be also characterized by reduction of the values of UV254, fluorescence intensity at 345 nm (Excitation)/435 nm (Emission) and that at 240 nm/435 nm to 0.06 /cm, 35 and 15, respectively.

The applicability of a sequential process of ozonation and ozone/hydrogen peroxide process for the removal of soluble organic compounds from a pre-coagulated municipal sewage was examined. 6-25% of initial T-CODCr was removed at the early stage of ozonation before the ratio of consumed ozone to removed T-CODCr dramatically increased. Until dissolved ozone was detected, 0.3 mgO3/mgTOC0 (Initial TOC) of ozone was consumed. When an ozone/hydrogen peroxide process was applied, additional CODCr was removed. And we elucidated that two following findings are important for the better performance of ozone/hydrogen peroxide process; those are to remove readily reactive organic compounds with ozone before the application of ozone/hydrogen peroxide process and to avoid the excess addition of hydrogen peroxide. Based on these two findings, we proposed a sequential process of ozonation and multi-stage ozone/hydrogen peroxide process and the appropriate addition of hydrogen peroxide. T-CODCr, TOC and ATU-BOD5 were reduced to less than 7 mg/L, 6 mgC/L and 5 mg/L, respectively after total treatment time of 79 min. Furthermore, we discussed the transformation of organic compounds and the removal of organic compounds. The removal amount of CODCr and UV254 had good linear relationship until the removal amounts of CODCr and UV254 were 30 mg/L and 0.11 cm-1, respectively. Therefore UV254 would be useful for an indicator for CODCr removal at the beginning of the treatment. The accumulation of carboxylic acids (formic acid, acetic acid and oxalic acid) was observed. The ratio of carbon concentration of carboxylic acids to TOC remaining was getting higher and reached around 0.5 finally. Removal of TOC was observed with the accumulation of carboxylic acids. When unknown organic compounds (organic compounds except for carboxylic acids) were oxidized, 70% was apparently removed as carbon dioxide and 30% was accumulated as carboxylic acids. A portion of biodegradable organic compounds to whole organic compounds was enhanced as shown by the increase ratio of BOD/CODCr.

It is shown that two knots can be transformed into each other by Cn-moves if and only if they have the same Vassiliev invariants of order less than n. Consequently, a Cn-move cannot change the Vassiliev invariants of order less than n and may change those of order more than or equal to n. In this paper, we consider the coefficient of the Conway polynomial as a Vassiliev invariant and show that a Cn-move changes the nth coefficient of the Conway polynomial by ±2, or 0. And for the 2mth coefficient (2m > n), it can change by p or p + 1 for any given integer p.

RNA/DNA ratio is a useful and reliable indicator of the nutritional status of fish larvae and juveniles. In order to assess the nutritional status of field-caught larval Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel), starvation experiments of hatchery-reared larvae were conducted and changes in the RNA/DNA ratio of fed and starved larvae were analyzed. Starvation experiments were conducted every 3 days after first feeding. The survival rate of Pacific bluefin tuna larvae ranged 10–50% after 1 day of starved conditions and growth retardation was observed immediately. These results suggest that Pacific bluefin tuna larvae have a very low tolerance to starvation. The RNA/DNA ratios of fed larvae were approximately 2.0–4.0. On the other hand, the value of starved larvae significantly decreased to 1.0–3.0. The nutritional status of 3 cohorts of field-caught tuna larvae collected in the northwestern Pacific Ocean was examined based on the value of the RNA/DNA ratio of the 1 day starved larvae. 4.35–25.77% of the cohorts were regarded as the “starving condition”, which was negatively correlated to the ambient prey densities. These findings suggest that the nutritional condition of larval Pacific bluefin tuna was influenced by the ambient prey density, and starvation itself and starvation-induced predation could greatly contribute to mortality in the larval period of Pacific bluefin tuna.

Young Pacific bluefin tuna Thunnus orientalis with archival tags were released in the East China Sea. Time-series data for depth, and ambient and peritoneal temperatures for nine fish, recorded every 128 s, were analyzed. Our objectives were to describe monthly changes in diving patterns in relation to the ambient thermal structure and the occurrence of feeding events during March–June, and to discuss possible reasons why bluefin tuna repeatedly dived to depths below the thermocline in terms of their thermoconservation mechanisms. It was found that the fish repeatedly dived through the thermocline at intervals of 1.2 h on average, and the dive frequency was high during March–May. However, the dive frequency and periodicity decreased in June, when the gradient became steeper. In contrast, feeding events increased in June. These results indicate that from March to May, bluefin tuna repeatedly dive because food biomass is inadequate at the surface, and they stop undertaking repeated dives in June when food becomes more readily available at the surface, in addition to low visibility caused by low solar radiation. Further, the range of heat transfer times for these fish was so long that their peritoneal temperature was probably maintained by engaging in brief dives. The periodicity of dives may lead to a lower fluctuation in the peritoneal temperature, suggesting that the dives are a kind of behavioral thermoregulation.

The effect of inorganic carbon on ozone self-decomposition was examined in this study. Experiments were carried out under a wide concentration range of inorganic carbon of 0–30 mgC/L. First, the rate constant in the overall description model with nth order reaction on dissolved ozone was evaluated. A clear difference in the rate constants with and without inorganic carbon was observed even when the concentration of inorganic carbon was as low as 3 mgC/L; the difference with and without inorganic carbon was almost one order of magnitude. Reaction rate constant was lower with an increase of inorganic carbon concentration. The reaction rate constants were in the range from 1.4 × 10−5–1.9 × 10−4 (mg/L)−1sec.−1 under pH of 6.8–8.1 and inorganic carbon concentration of 6–30 mgC/L and converged on the order of 1.0 × 10−4 (mg/L)−1sec.−1. The difference of the second-order reaction rate constant under the practical range of pH and inorganic carbon concentration was one order of magnitude at most. Depression of ozo...

Recently, due to the advancement of O3/H2O2 treatment processes, it has been necessary to further develop the ozone self-decomposition radical model, which includes a variety of radical reactions, because the hydroxyl radical concentration as well as the ozone concentration must be taken into consideration. In this study, an ozone self-decomposition model that is meaningful for environmental engineering design under the practically encountered pH condition in the time scale of water treatment is proposed. First, an overall description model of ozone self-decomposition is developed based on the experimental results. The evaluation clarifies that the ozone self-decomposition can be formulated by a second-order reaction and the rate constant is enhanced 5 times and 2.2 times with increase of one pH unit and 5°C increase of temperature, respectively. Then a radical reaction model was developed. All of the reaction rate constants included in the model are obtained from the literature review except for the reac...

A simple model is developed to predict the formation of bromate ion as well as hypobromous acid/hypobromite ion through the hydroxyl radical pathway. For simplicity of the model, hydroxyl radical concentrations are represented by the concentration ratio of hydroxyl radical to dissolved ozone under the different pH conditions. A kinetic analysis is conducted to evaluate the ratio under the different pH conditions based on the experimental data. The different extent of the ratio by one pH unit is found to be 3–4 times. This model can favorably simulate the formations of bromate ion as well as hypobromous acid/hypobromite ion in spite of the simplicity of the model. So it is likely that this model will be applicable to the prediction of bromate ion formation in water purification process such as drinking water treatment by introducing the concentration ratio of hydroxyl radical to dissolved ozone.

The purposes of this study are to investigate ozonation characteristics, to evaluate oestrogenicity and to confirm behaviours of by-products during ozonation of secondary effluent. For the ozonation of secondary effluent, TOC was decreased only 10% when 4 (mgO3/mgC) of ozone consumption per initial TOC. However, UV254 and SUVA was decreased approximately 65% until ozone consumption of 2 (mgO3/mgC). Ozonation was also shown to be very effective for decrease of oestrogenicity in secondary effluent. Bromate ion started to form obviously when the ozone consumption per initial TOC exceeded 2 (mgO3/mgC) and increased while the time of ozonation became longer. From these results, ozone consumption per initial TOC was shown to be an appropriate operation parameter to reduce SUVA effectively and E2 equivalent concentration to less than 0.1 nM without significant formation of bromate ion, and its value was determined to be 2 (mgO3/mgC) in ozonation of secondary effluent.

This study aims to establish appropriate conditions to control the formation of bromate ion and brominated organic compounds during the O3/H2O2 treatment of the secondary effluents of sewage. When the H2O2/O3 mole ratio of injection was above 0.5 and the dissolved ozone concentration was below 0.1 mg/L, bromate ion formation was controlled, and treatment purposes such as the reduction of estrogenic activity or organic matter were completed. The formation of TOBr and individual brominated organic compounds during O3/H2O2 treatment was also completely controlled.

Immature Pacific bluefin tuna Thunnus orientalis, tagged with archival tags, were released near Tsushima Island in the East China Sea (ECS) during the winters of 1995, 1996 and 1997. Geolocations were estimated using the archival tags from recovered fish. These data, together with sea surface temperature (SST) data from satellite remote sensing, are used to describe the habitat used by these bluefin in the ECS from January to June for 3 years (1996, 1997, 1998), and to asses the effect of water temperature on fish distribution and movement. The results indicate that their geolocations ranged from the area north-east of Tsushima Island to the offshore area in the southwest. However, the area of highest density differed among years, being furthest south in 1996 and furthest north in 1998. The differences were probably caused by changes in SST associated with La Nina (1996) and EI Nino (1998) events. Another densely populated area was identified in offshore waters of latitude 28–30 oN in 1996 (only), on the cold side of the Kuroshio front. These fish may have been prevented from moving northwards by an intrusion of Kuroshio water of approximately 25°C into the region immediately to the north-east.

Treatment experiments by two laboratory scale semi-batch reactors and a demonstration scale process were both carried out to evaluate endocrine disrupting chemical removal characteristics by ozonation in sewage treatment. In the semi-batch mode experiments, secondary effluent and primary effluent of sewage treatment plants were ozonated respectively. Behaviour of brominated by-products was also evaluated. The results based on these studies indicated that estrogenicity in wastewater can be removed by ozonation effectively. The condition of ozonation until dissolved ozone concentration increased to 0.1 mg/L, which corresponded to 1 mgO3/mgC and 0.4 mgO3/mgC of ozone consumption per initial DOC for secondary effluent and primary effluent, respectively, is proposed as an appropriate operational condition for efficient removal of EDCs as well as UV254 and SUVA without production of brominated by-products.

To estimate the survival process of Pacific bluefin tuna Thunnus orientalis during the larval period, estimated growth histories were compared between larvae collected in late spring and juveniles collected in the boreal summer of 2004, which were considered to be survivors of the larval cohorts. Larval tuna (3.3 to 9.6 mm standard length, SL) were collected from mid-May to early June around the Ryukyu Islands, northwestern Pacific Ocean, and juvenile tuna were collected offshore of Kochi and Nagasaki prefectures in July-August. Preflexion, flexion and postflexion larvae were col- lected, and their ages ranged from 4 to 18 d. Back-calculated SLs by the biological intercept method showed that larval tuna in the postflexion phase were larger-at-age than preflexion and flexion larvae, suggesting that only larger and faster growing larvae were able to survive to the postflexion phase. The logarithms of otolith radii (ln OR: proportional to SL) of larvae with slower growth and development were smaller than the minimum ln OR of surviving juvenile tuna, which indicated the smallest possible size required for larvae to successfully recruit to the fishery. These results indicate that the survival of larvae of Pacific bluefin tuna depends largely on size and growth rates during early life history.

The recruitment abundance index of Pacific bluefin tuna Thunnus orientalis was estimated from 1980 to 2003 fishing year by using the troll fishery data in Nagasaki Prefecture, western Japan. It has been shown that the troll fishery in Nagasaki Prefecture operates with good time-area coverage of the species habitat, and that the fishing power slightly changed during the period analyzed, based on fisheries statistics, published information, and interviews with the fishers. Average catch per unit effort (CPUEs) were standardized by a generalized linear model (GLM) considering the effects of fishing year, season and landing area. Standardized CPUE of age-O bluefin tuna showed larger fluctuations year by year than the nominal CPUE combined for all ages. High CPUEs in fishing years of 1981, 1994, 1996 and 1999 were observed. Data from these years agreed with the higher recruitments estimated by virtual population analysis (VPA) or higher catch of age-O fish reported for the Pacific side. The age-specific standardized CPUE of age-O bluefin tuna in this study was judged to be a useful indicator of recruitment.

Ozonation and ozone process combined with hydrogen peroxide have been identified as new technologies for direct or indirect reuse of wastewater. This study aims to establish appropriate conditions to control the formation of BrO 3 - and brominated organic compounds during O 3 /H 2 O 2 treatment of secondary effluents of sewage. When the H 2 O 2 /O 3 mole ratio of injection was above 0.5 and the DO 3 concentration was below 0.1 mg/L, BrO 3 - was controlled as well as treatment purpose was completed. TOBr formation in O 3 /H 2 O 2 treatment was also completely controlled.

Immature Pacific bluefin tuna Thunnus orientalis, tagged with archival tags, were released near Tsushima Island in the East China Sea during the winters of 1995 through 1998. Time-series data for ambient and peritoneal cavity temperatures, recorded every 128 or 256 s for 23 fish recovered, were analyzed. The objective of this study was to clarify the process of development of thermoconservation ability with growth in relation to adaptive mechanisms to cooler temperate waters. According to the results, mean ambient temperatures ranged from 14.9 to 20.7°C, which is almost within the optimum temperature range according to previous reports. Mean peritoneal temperatures were higher than ambient temperatures (19.7–27.3°C), but never reached 35°C, which would induce overheating. Although the mean thermal differences between peritoneal and ambient temperatures increased with body size, the rate of increase decreased with body size. A heat budget model suggests that as the insulation of the body develops, the estimated mean values of internal heat production decrease with body size. This is probably due to the allometric scale effect and explains why the thermal difference does not increase quickly with body size. It is likely that Pacific bluefin tuna inhabit cooler temperate waters in mid-latitude regions to avoid overheating.

[Nucleotide sequence analysis of the mitochondrial COI gene was performed to identify midto final stage phyllosoma larvae of spiny lobsters of the genus Panulirus caught in the south of the Ryukyu Archipelago (northwest Pacific). The identified larvae were subjected to morphological investigation. All 92 larvae caught in May 2003 were late to final stage phyllosomas of P. japonicus, while 174 larvae in November 2004 comprised four species of phyllosoma group 1 (P. femoristriga (n = 1), P. japonicus (4), P. longipes bispinosus (110), and P. l. longipes (7)), one of group 2 (P. penicillatus (43)), and two of group 4 (P. ornatus (8) and P. versicolor (1)). Photo images of representative phyllosoma larvae, substantiated by molecular identification, are presented. Morphological investigation indicated that the ratio between the widths of cephalon and thorax (CT ratio) of the P. japonicus phyllosomas was the smallest, and discrimination of P. japonicus larvae from the other species of the "P. japonicus group" may be possible using the CT ratio combined with the smaller body size. Intraspecific variation was observed in the arrangement of subexopodal spines of P. ornatus and P. versicolor phyllosomas, indicating this character is not diagnostic to separate these two species. L'analyse de la sequence nucleotidique du gene mitochondrial COI a ete realisee afin d'identifier les larves phyllosomes de langoustes du genre Panulirus, aux stades larvaires allant du milieu a la fin du developpement, collectees au sud de l'archipel des Ryukyu (Pacifique nord-occidental). Les larves identifiees, ont ete soumises a un examen morphologique. Les 92 larves collectees en mai 2003 etaient des phyllosomes du dernier stade de P. japonicus, tandis que les 174 larves de novembre 2004 comprenaient quatre especes de phyllosomes de groupe 1 (P. femoristriga (n = 1), P. japonicus (4), P. longipes bispinosus (110) et P. l. longipes (7)), une du groupe 2 (P. penicillatus (43)) et deux du groupe 4 (P. ornatus (8) et P. versicolor (1)). Des images photo des larves phyllosomes representatives, corroborees par l'identification moleculaire, sont presentees. L'analyse morphologique a montre que le rapport entre les largeurs du cephalon et du thorax (rapport CT) des phyllosomes de P. japonicus etait le plus bas, et la separation des larves de P. japonicus des autres especes du «groupe P. japonicus» peut etre faite en utilisant le rapport CT combine avec la taille plus petite du corps. Une variation intraspecifique a ete observee dans l'arrangement des epines subexopodiales des phyllosomes de P. ornatus et de P. versicolor, ce qui indique que ce caractere n'est pas diagnostique pour separer ces deux especes., Nucleotide sequence analysis of the mitochondrial COI gene was performed to identify midto final stage phyllosoma larvae of spiny lobsters of the genus Panulirus caught in the south of the Ryukyu Archipelago (northwest Pacific). The identified larvae were subjected to morphological investigation. All 92 larvae caught in May 2003 were late to final stage phyllosomas of P. japonicus, while 174 larvae in November 2004 comprised four species of phyllosoma group 1 (P. femoristriga (n = 1), P. japonicus (4), P. longipes bispinosus (110), and P. l. longipes (7)), one of group 2 (P. penicillatus (43)), and two of group 4 (P. ornatus (8) and P. versicolor (1)). Photo images of representative phyllosoma larvae, substantiated by molecular identification, are presented. Morphological investigation indicated that the ratio between the widths of cephalon and thorax (CT ratio) of the P. japonicus phyllosomas was the smallest, and discrimination of P. japonicus larvae from the other species of the "P. japonicus group" may be possible using the CT ratio combined with the smaller body size. Intraspecific variation was observed in the arrangement of subexopodal spines of P. ornatus and P. versicolor phyllosomas, indicating this character is not diagnostic to separate these two species. L'analyse de la sequence nucleotidique du gene mitochondrial COI a ete realisee afin d'identifier les larves phyllosomes de langoustes du genre Panulirus, aux stades larvaires allant du milieu a la fin du developpement, collectees au sud de l'archipel des Ryukyu (Pacifique nord-occidental). Les larves identifiees, ont ete soumises a un examen morphologique. Les 92 larves collectees en mai 2003 etaient des phyllosomes du dernier stade de P. japonicus, tandis que les 174 larves de novembre 2004 comprenaient quatre especes de phyllosomes de groupe 1 (P. femoristriga (n = 1), P. japonicus (4), P. longipes bispinosus (110) et P. l. longipes (7)), une du groupe 2 (P. penicillatus (43)) et deux du groupe 4 (P. ornatus (8) et P. versicolor (1)). Des images photo des larves phyllosomes representatives, corroborees par l'identification moleculaire, sont presentees. L'analyse morphologique a montre que le rapport entre les largeurs du cephalon et du thorax (rapport CT) des phyllosomes de P. japonicus etait le plus bas, et la separation des larves de P. japonicus des autres especes du «groupe P. japonicus» peut etre faite en utilisant le rapport CT combine avec la taille plus petite du corps. Une variation intraspecifique a ete observee dans l'arrangement des epines subexopodiales des phyllosomes de P. ornatus et de P. versicolor, ce qui indique que ce caractere n'est pas diagnostique pour separer ces deux especes.]

To resolve the controversial taxonomic status of two closely-related lanternfishes, Diaphus perspicillatus (Ogilby, 1898) and D. gigas Gilbert, 1913, mitochondrial cytochrome b (cyt b) gene sequences of representative individuals were obtained, and specifically-diagnostic restriction sites investigated. In the 5 -half nucleotide sequences of the cyt b gene (594 bp), a relatively high level of nucleotide substitution between the species (8.2–8.7%), together with very low intraspecific variation ( 0.86%), was observed, which clearly indicates the two morphs to be specifically distinct. Previous hypotheses regarding gigantism of D. gigas and suspicions regarding the synonymy of the two species are rejected. The nucleotide sequences and practical restriction enzyme assay indicate that any one of four restriction endonucleases (Fok I, Hae III, Nla IV, and Rsa I) can unambiguously discriminate between the two species. Based on specimens identified by molecular analysis, eye diameter and gill raker count were found to be good diagnostic morphological characters.

The contribution of ozone and hydroxyl radical to the formation of bromate ion was investigated in a continuous flow reactor. Experiments were conducted under a wide range of ozone dose (0.7 ∼ 3.8 mgL), pH (6.5 ∼ 8.5), and t-butanol concentration (0 ∼ 0.5 mM). The formation of bromate ion was found to depend on radical reaction pathway, because the amount of bromate ion formed increased with pH and decreased with t-butanol, a radical scavenger, even when dissolved ozone concentrations were almost the same. In fact, the amount of bromate ion formed was reduced by 90% in the presence of t-butanol. Furthermore, the formation of bromate ion occurred even when dissolved ozone was not significantly detected in the presence of organic matter (TOC of 1 mgCL). The second-order reaction rate constant of hydroxyl radical with bromide ion, k HO,Br− of 1.7 × 109 (M−1s−1), was obtained on the assumption that the reactions of bromide ion and t-butanol with hydroxyl radical were competitive with each other in th...

To evaluate the decomposition and formation characteristics of estrogenic compounds during ozonation, semibatch ozonation experiments were conducted using the prototypical estrogen, 17β-estradiol (E2). Ozonation was shown to be very effective for decomposition of E2 and decrease of estrogenicity. An E2 equivalent concentration index was introduced based on the estrogenicity intensity measured with Estrogen Competition Binding Assay (ECB Assay). The importance of evaluation of estrogenicity by ECB Assay was shown by the difference in removal efficiencies between E2 equivalent concentration and the E2 itself. Through this research, ozonation was shown to be a promising method to remove E2 without any formation of estrogenic by-products from secondary-treated sewage, which contained E2 of ngL level and total organic carbon (TOC) of several mgL.