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Experiments to characterize retained sludge in a down-flow hanging sponge (DHS) reactor fed with upflow anaerobic sludge blanket (UASB) treated sewage under moderate conditions were conducted. Plenty of oxygen was supplied through the DHS reactor without aeration and the effluent qualities after the reactor were comparable to activated sludge processes. The average excess sludge production rate was 0.09 g SS g −1 COD removed. The DHS reactor maintained a high sludge concentration of 26.9 g VSS L −1 sponge, resulting in a low loading rate of 0.032 g COD g −1 VSS day −1 . The endogenous respiration rate of DHS sludge was comparable to previously reported aerobic sludges. The numbers of microfauna were one order of magnitude greater than those in activated sludge. The results indicated that low excess sludge production was attributable to the high sludge concentration, sufficient oxygen supply, adequate endogenous respiration rate, and a high density and diversity of microfauna.

Sewage treatment processes can be considered as artificial ecosystems, in which eukaryotes are key microbial components. Protozoans and metazoans are often used as indicators of treatment performance. Eukaryotes are the primary predators of prokaryotes; their predation of dispersed bacteria improves sludge sedimentation and effluent water quality in terms of turbidity, biological oxygen demand (BOD), and suspended solids (27), and also decreases the risk of exposure to bacterial pathogens (28). Some Rotifera lineages specifically graze on filamentous fungi that are known to cause bulking in activated sludge (10), and these organisms can, therefore, be used to prevent sludge bulking. Furthermore, recent studies reported the useful implementation of fungi for granular formation in sewage treatment processes (35) as well as the degradation of cellulose, hemicellulose, and lignin biomass (7). Fungi are known to contribute to denitrification, and some can grow under various O2 conditions (concentrations) through three different energy-yielding metabolic pathways: O2 respiration, denitrification (nitrite respiration), and ammonia fermentation (13). Some eukaryotes belonging to the Rhizaria have been reported to accumulate nitrate under aerobic conditions and to respire using this accumulated nitrate through denitrification under anoxic conditions (31). Due to these important processes, the contribution of eukaryotes to denitrification in ocean sediments is estimated to be equal to that of bacteria (29); thus, eukaryotes may also play a significant role in the removal of carbon and nitrogen in sewage treatment processes. The eukaryotes involved in sewage treatment processes have traditionally been identified morphologically and enumerated by microscopic observations. However, morphological identification is often hampered due to limited or difficult diagnostic criteria for some taxa and the time-consuming processes necessary to acquire identification expertise. Minidiscus cells are easily overlooked because of their small size (1.9–7.5 μm) (25). Furthermore, the functional identification of eukaryotes requires that they are isolated, which can be difficult, especially for small eukaryotes. Thus, the role played by eukaryotic species in many environments—including wastewater treatment processes—remains unclear. These limitations have driven the development of alternative (i.e., molecular) identification methods. The phylogenetic diversity of eukaryotes has been investigated by the construction of 18S rRNA gene clone libraries or tag pyrosequencing methods (3, 25, 33). The molecular diversity of eukaryotes in activated sludge sewage treatment processes has also been investigated (8, 9, 23, 26, 27, 36). These studies investigated specific members of eukaryotes (e.g., Ciliophora and Fungi) or eukaryotes in lab-scale reactors with a small number of clones. Thus, there have been no comprehensive investigations of eukaryotic molecular diversity in full-scale sewage treatment processes, and the diversity and roles of eukaryotes there consequently remain unclear. Eukaryotic communities in sewage treatment processes are influenced by the type of process and operating conditions (6). Various processes are employed to treat sewage, and each full-scale sewage treatment plant receives different amounts and concentrations of sewage, resulting in different operational conditions. Therefore, the eukaryotic compositions of full-scale sewage treatment plants are expected to be highly diverse, and previously unrecognized eukaryotes may be present and play important roles. We here investigated the eukaryotic communities of 9 sludge samples collected from 3 different full-scale sewage treatment processes (activated sludge [AS], anoxic/oxic activated sludge [AO], and oxidation ditch [OD]) by constructing 18S rRNA gene clone libraries. We identified core and shared eukaryotes in sewage treatment processes. Furthermore, the results obtained showed that sewage treatment processes can be characterized by a greater diversity of uncultured eukaryotes than was previously recognized.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as sulindac and indomethacin inhibit colon carcinogenesis, and selective cyclooxygenase (COX)-2 inhibitors are considered to be potential chemopreventive agents without the side effects of usual NSAIDs. We reported that NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide, suppressed the formation of preneoplastic lesions, aberrant crypt foci (ACF), induced by azoxymethane (AOM) in a short-term assay of rat colon carcinogenesis. In this study, we examined the effects of long-term NS-398 administration on rat colon carcinogenesis. After three AOM treatments at weekly intervals, a dose of 10 mg/kg of NS-398 in 5% Arabic gum solution was administered by gavage three times per week in group 2 until the termination of the experiment. Rats in group 1 were fed in a basal diet and given 5% Arabic gum solution alone after AOM treatment. At 40 weeks after the first AOM treatment, all rats were killed and the whole intestines including colon were examined. While the incidences of whole intestinal and colon neoplasms in group 1 were 84.6% and 80.8%, respectively, those in group 2 (given NS-398) were 51.9% and 44.4% respectively (P=0.0177 and P=0.0103 by Fisher's exact test, respectively). The multiplicities in group 2 (0.67 ± 0.78 and 0.48 ± 0.58) were also decreased significantly compared with those (1.39 ± 1.10 and 1.08 ± 0.74) in group 1 (P < 0.01 by Welch's method and P < 0.002 by Student's t test, respectively). In immunohistochemistry for proliferative cell nuclear antigen (PCNA), the PCNA-stained cell index (7.40 ± 0.5) in group 2 was significantly decreased from that in group 1 (14.03 ± 0.82) (P < 0.001 by Welch's method). The results suggest that NS-398, a selective COX inhibitor, has a chemopreventive activity against colon carcinogenesis without side-effects such as gastric ulceration.

Modifying effects of dietary exposure of protocatechuic acid (PCA), a natural monophenolic compound, S-methylmethanethiosulfonate (MMTS), an organosulfur compound newly isolated from cauliflower, and 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone (KYN-54), a novel retinoidal butenolide compound, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (10 micromol, [corrected] single i.p. injection)-induced pulmonary carcinogenesis were examined in female A/J mice. Each of the test chemicals was given in diets during initiation or post-initiation phases (PCA, 1000 ppm; MMTS, 100 ppm; KYN-54, 200 ppm). All of these which had been proved to be chemopreventive mainly in digestive-organs carcinogenesis did not exert any preventive effect in this model when the incidence or multiplicity of pulmonary tumors (adenomas) of mice given NNK and the test chemical at the termination of the experiment (4 months) was compared to that of mice exposed to the carcinogen alone. In contrast, the multiplicity of lung tumors of mice receiving KYN-54 during the post-initiation phase was significantly larger than of the animals with NNK alone (P0.05), showing that KYN-54 has a promoting effect on pulmonary carcinogenesis in mice. These data indicate an organotropic activity of these compounds and suggest that candidate compounds for cancer chemoprevention need to be carefully examined for effectiveness in multiple organs by different models.

The modifying effects of citrus auraptene given during the initiation and post-initiation phases of oral carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. At 6 weeks of age, animals were divided into experimental and control groups, and fed the diets containing 100 ppm or 500 ppm auraptene. At 7 weeks of age, all animals except those treated with auraptene alone and control groups were given 4-NQO (20 ppm) in the drinking water for 8 weeks to induce tongue carcinoma. Starting 7 days before the 4-NQO exposure, groups of animals were fed the diets containing auraptene (100 and 500 ppm) for 10 weeks and then switched to the basal diet. Starting 1 week after the cessation of 4-NQO exposure, the groups given 4-NQO and a basal diet were switched to the diets mixed with auraptene (100 and 500 ppm), and maintained on these diets for 22 weeks. The other groups consisted of rats fed auraptene alone (500 ppm) or untreated rats. All rats were necropsied at the termination of the study (week 32). The incidences of tongue lesions (neoplasms and preneoplasms), polyamine levels in the tongue tissue and cell proliferation activity estimated by 5-bromodeoxyuridine (BrdU)-labelling index were compared among the groups. In addition, the activities of gluthathione S-transferase (GST) and quinone reductase (QR) in liver and tongue of rats gavaged various doses of auraptene (0, 200, 400 and 800 mg/kg body wt) for 5 days were assayed. Feeding of auraptene at both doses during the initiation phase caused a significant reduction in the frequency of tongue carcinoma (100 ppm auraptene, 91% reduction, P < 0.001; 500 ppm auraptene, 63% reduction, P < 0.05). When fed auraptene after 4-NQO exposure, the frequency of tongue carcinoma was also decreased (100 ppm auraptene, 100% reduction, P < 0.001; 500 ppm auraptene, 74% reduction, P < 0.01). The incidences of tongue severe dysplasia in these groups were significantly smaller than those in carcinogen controls (P < 0.05). There were no pathological alterations in rats treated with 500 ppm auraptene alone or those in an untreated control group. Dietary administration of auraptene significantly decreased BrdU-labelling index and polyamine concentrations in the oral mucosa (P < 0.05). In addition, auraptene administration significantly increased the activities of GST and QR in the liver and tongue. Although dose-dependent effect was not found, citrus auraptene is effective in inhibiting the development of oral neoplasms induced by 4-NQO. Thus, suppression by the initiation-feeding of auraptene might relate to elevation in the phase II enzymes GST and QR of the liver and tongue, and inhibition occurring during the post-initiation might be related to suppression of increased cell proliferation caused by 4-NQO in the oral mucosa.

We investigated the modifying effects of nabumetone, a relatively selective cyclooxygenase-2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N-methyl-N-nitrosourea(MNU)-induced mammary carcinogenesis in female Sprague-Dawley rats. A total of 124 rats, 6 weeks old, were divided into 6 groups. At 50 days of age, groups 1, 2, and 3 were treated with MNU (50 mg/kg body weight) by subcutaneous injection. From the age of 8 weeks, groups 2 and 4 were given 0.03% nabumetone in the diet and groups 3 and 5 were given 0.03% esculetin in the diet. All rats were necropsied at the termination (25 weeks after the start of experiment). The incidence and multiplicity of neoplasms in group 2 were significantly smaller than those in group 1 (P < 0.005 and P < 0.001, respectively). The incidence of neoplasms in group 3 was also significantly smaller than that in group 1 (P < 0.05). These results indicate that the intake of nabumetone or esculetin during the time corresponding to the post initiation phase has a chemopreventive effect on MNU-induced mammary carcinogenesis in rats.

Prostaglandin E2, which is produced by cyclooxygenase (COX) during arachidonic acid metabolism, is considered to be related to colon Carcinogenesis. Therefore, the effect of NS-398 (N-(2-cyclohexyl-oxy-4-nitrophenyl)methanesulfonamide), a COX-2 inhibitor, was examined in azoxymethane (AOM)-induced colon Carcinogenesis in rats in this study. In the first experiment, groups 1–3 were treated with AOM (15 mg/kg, s.c.) 3 times at intervals of a week from 5 weeks of age. Groups 2 and 3 were respectively given 1 mg/kg and 10 mg/kg of NS-398 in 5% gum arable aqueous solution 3 times per week by oral garage during the experiment. Six weeks after the first exposure to AOM, aberrant crypt foci (ACF) were counted in the colonic mucosa of all rats. The mean occurrence of ACF per length in rats given 1 mg/kg b.w. or 10 mg/kg b.w. of NS-398 was reduced to 65.7% or 52.8%, respectively, of that in rats treated with only AOM. Levels of COX-2 mRNA expression in groups treated with AOM, regardless of NS-398, were slightly higher than that in the group treated with NS-398 alone as judged from reverse transcription-polymerase chain reaction analysis. In the second experiment, the effect of NS-398 at different times, i.e., during initiation and post-initiation, was examined. Treatment with NS-398 in both phases significantly inhibited the appearance of ACF. The results imply that NS-398 might have a chemopreventive potential.

Wistar rats exposed to inescapable foot shocks (IS) for 2 wk exhibited PTSD-like bi-directional changes similar to avoidance/numbing and hyperarousal symptoms when placed in a shuttle box. Paroxetine administration after IS reduced the hyperarousal-like behavior, and its therapeutic effect on avoidance/numbing-like behavior was also significant. Further, F344 rats, which were more vulnerable to various kinds of stressors, showed more significant 'bi-directional changes' than Wistar rats. Thus, the paradigm we have developed could serve as a useful PTSD model because of its face, predictive, and construct validity. Moreover, the intensity of IS dose-dependently induced PTSD-like behaviors and hypo-activity in a shuttle box, similar to the 'avoidance/numbing' that reappeared in a square open field. These findings further support the construct validity of this paradigm. Both electro-convulsive shock treatment before and after IS ameliorated the PTSD-like behaviors in this model, so electro-convulsive therapy may be an effective method for prevention and medical treatment of PTSD in the future. On the other hand, pretreatment with fluvoxamine before IS did not have a significant effect, and its improving effect after IS was only observed for 'hyperarousal' behavior. Lastly, we recently developed a useful criterion, which is represented as a 'bi-directional index', for separating real PTSD rats from those exposed to IS.

Polyphenolic compound chlorogenic acid (CGA) known to be much contained in coffee beans was found to have a regressive effect on induced aberrant crypt foci (ACF) as well as on development of ACF in azoxymethane (AOM)-induced colorectal carcinogenesis in rats. Rice germ and gamma-aminobutyric acid-enriched defatted rice germ inhibited AOM-induced ACF formation and colorectal carcinogenesis in rats. Ferulic acid (FA) also known to be contained in coffee beans and rice prevented AOM-induced ACF formation and intestinal carcinogenesis in rats. Both of food factors, coffee and rice may be of benefit to prevention of human colorectal cancers.

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta- d -glucoside, is one of xanthone derivatives and C-glucosylxanthones, is widely distributed in higher plants and is one of constituents of folk medicines. Recent studies showed that mangiferin has a potential as an anti-oxidant and an anti-viral agent. In this study, we examined the effects of mangiferin in rat colon carcinogenesis induced by chemical carcinogen, azoxymethane (AOM). We performed two experiments: a short-term assay to investigate the effects of mangiferin on the development of preneoplastic lesions by AOM, aberrant crypt foci (ACF), and the following long-term assay for the influence of mangiferin on tumorigenesis induced by AOM. In the short-term assay, 0.1% mangiferin in a diet significantly inhibited the ACF development in rats treated with AOM compared to rats treated with AOM alone (64.6±22.0 vs. 108.3±43.0). In the long-term assay, the group treated with 0.1% mangiferin in initiation phase of the experimental protocol had significantly lower incidence and multiplicity of intestinal neoplasms induced by AOM (47.3 and 41.8% reductions of the group treated with AOM alone for incidence and multiplicity, respectively). In addition, the cell proliferation in colonic mucosa was reduced in rats treated with mangiferin (65–85% reductions of the group treated with AOM alone). These results suggest that mangiferin has potential as a naturally-occurring chemopreventive agent.

The modifying effects of dietary administration of ferulic acid (FA) on azoxymethane (AOM)-induced colon carcinogenesis were examined in three experiments with male 344 rats. In the first experiment, the modifying effect of FA on AOM (15 mg/kg body weight, once a week, for 3 weeks)-induced formation of aberrant crypt foci (ACF) was examined in five groups. Numbers of ACF/colon of groups 2 (AOM+250 ppm FA) and 3 (AOM+500 ppm FA) at the termination (5 weeks after the start) were smaller than of group 1 (AOM alone). Those of ACF/cm(2) of group 3 were also smaller than of group 1 (P

The modifying effects of chlorogenic acid (CA) on N-methyl-N-nitrosourea (MNU)-induced glandular stomach carcinogenesis were investigated in five groups of male F344 rats. Rats in Groups 1 through 3 were given MNU in drinking water at a concentration of 400 ppm for 12 weeks. Animals of Group 1 were then kept on the basal diet alone, and those of Group 2 or 3 were fed a diet containing 500 or 250 ppm CA for a subsequent 22 weeks. Group 4 was exposed to CA alone through the experimental period (36 weeks), and Group 5 was given the basal diet continuously and treated as a control. At the end of the experiment, the incidence of glandular stomach carcinoma of Group 3 was significantly smaller than that of Group 1 (p < 0.03). The incidence of adenomatous hyperplasia of Group 2 was also significantly lower than that of Group 1 (p < 0.02). In addition, the proliferating cell nuclear antigen (PCNA) labeling index of the epithelial cells from the non-neoplastic mucosa in rats of Group 2 or 3 was significantly smaller than that of Group 1 (p < 0.0001). These results suggest that CA has a chemopreventive effect on MNU-induced rat glandular stomach carcinogenesis by exposure during the post-initiation phase, and CA may be a promising agent for prevention of human stomach cancer.

The present study was designed to investigate the effect of Fas-mediated liver cell apoptosis, induced by a hamster monoclonal antibody against mouse Fas antigen, on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. DEN (10 μg g–1, intraperitoneally (i.p.)) was given to 15-day-old male C3H/HeJ mice. Three weeks after DEN treatment, Fas-mediated liver cell apoptosis induced by anti-Fas antibody resulted in a biphasic effect on induction of liver cell tumours, depending on dosage and time of antibody administration. Single or multiple treatment with high dose anti-Fas antibody (5 μg animal−1), induced gross liver cell damage and decreased the incidence of liver cell tumours in DEN-treated mice. In contrast, five treatments with low dose anti-Fas antibody (2 μg animal−1), induced dispersed localized liver cell damage and promoted the number of large-sized liver cell adenomas and hepatocellular carcinomas. These findings suggest that high dose anti-Fas antibody has a marked effect on the clearance of DEN-initiated liver cells, whereas repeated administration of low dose anti-Fas antibody promotes hepatocarcinogenesis. It is concluded that Fas-mediated liver cell apoptosis has a biphasic effect on hepatocarcinogenesis. © 2000 Cancer Research Campaign

The modifying effect of dietary administration of defatted rice-germ and gamma-aminobutyric acid (GABA)-enriched defatted rice-germ on azoxymethane (AOM)-induced colon carcinogenesis was investigated in two experiments with male F344 rats. In the first experiment (the pilot study), the effects of the defatted rice-germ, the GABA-enriched defatted rice-germ and rice-germ on AOM-induced (15 mg/kg body wt once a week for 3 weeks) formation of aberrant crypt foci (ACF) were examined. The latter two preparations (2.5% in the diet) significantly inhibited ACF formation (P < 0.005). In the second experiment, a long-term study of the effects of rice-germ was done. One group was treated with AOM alone, four groups received the carcinogen and were fed the diets containing 2.5% rice-germ or 2.5% GABA-enriched defatted rice-germ for 5 (initiation phase) or 30 weeks (post-initiation phase), two groups were treated with rice-germ or GABA-enriched defatted rice-germ alone and one group was kept on the basal diet. At the termination of the study, dietary exposure to rice-germ during the initiation phase significantly reduced the incidence of colonic adenocarcinoma (71 versus 29%, P < 0.01). GABA-enriched defatted rice-germ or rice-germ during the post-initiation phase also decreased the frequency of colonic adenocarcinoma (71 versus 20%, GABA-enriched defatted rice-germ feeding, P < 0.01; 27%, rice-germ feeding, P < 0.01). These data suggest that constituents of rice-germ are possible dietary preventatives for human colon cancers.

Alterations in multiple phosphorylation sites on exon 3 of the beta-catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical-induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the beta-catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction-single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the beta-catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN-induced hepatocarcinogenesis in rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines derived from cooked meat. In long-term experiments using rodents, carcinogenicity of PhIP in colon, mammary gland and prostate has been demonstrated. In this study, an experiment was designed to determine the apoptosis-inducing capacity of PhIP in colonic epithelium, a target organ for PhIP carcinogenicity, and possible modulating effects of beta-naphthoflavone (beta-NF), a P4501A2 inducer, on the apoptosis in rats. Out of eight groups of male F344 rats, four were given beta-NF in diet (1000 ppm) for a week beginning at 5 weeks of age. Four groups were given PhIP (100 mg/kg body weight) by gavage at 6 weeks of age. Twenty-four hours after the dosing of PhIP, cell death with typical morphology of apoptosis was apparent in the colon and the apoptotic index was significantly greater (P0.01) than of the control rats without exposure to PhIP. Prior administration of beta-NF caused significant acceleration of the induction of apoptosis by PhIP. Since PhIP requires metabolic activation by P4501A2 to exert genotoxic activities, the modulating effect of beta-NF on the PhIP-induced apoptosis will be through a P4501A2-dependent mechanism. Such assay of apoptotic indices in the colon may be useful not only for the evaluation of genotoxicity and/or the initiating capability of chemical agents with potentials for colorectal cancer, but also for the analysis of modifying agents on the carcinogenesis in the large bowel.

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)-induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM-induced colon tumorigenesis when it was fed to rats during the initiation or post-initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15 mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post-initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P = 0.03 and P = 0.001, respectively). The post-initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P = 0.06 and P = 0.00003, respectively). Such inhibition was dose-dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM-induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.