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Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23μM and 2.48±0.02μM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A new inhibitor of TNF-α production (IC50=0.89 μM) named vialinin C (1) was isolated from dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis. The structure of 1 was determined by high-resolution MS, NMR spectroscopic analysis, and confirmed by synthesis. Synthesis of ganbajunin B (5) obtained from the same origin was also described., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Vialinin A is an extremely potent inhibitor of tumor necrosis factor (TNF)-α release from RBL-2H3 cells. The present study investigated in detail the inhibitory effects of vialinin A and its analog, 5',6'-dimethyl-1,1':4',1″-terphenyl-2',3',4,4″-tetraol (DMT), on TNF-α. Vialinin A and DMT inhibited the release of TNF-α from RBL-2H3 cells in a dose-dependent manner, but had no effect on β-hexosaminidase activity. Also, vialinins had little effect on TNF-α mRNA levels. Intriguingly, vialinins inhibited TNF-α production at low concentrations, but not shown a dose-dependency. The potent inhibitory activities of vialinins against TNF-α production and release suggest promising new candidate pathways for anti-inflammatory agents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 µM for compound 1 and 3.3 ± 0.2 µM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m/z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC₅₀ of 20.3 µM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²⁺ influx from an internal store in RBL-2H3 cells.

A highly polymethylated flavone that effectively inhibited cytochrome P450s (CYPs) 1A2 and 3A4 (IC(50) = 2.41 and 1.71 µM) in vitro was isolated from thyme leaves (Thymus saturoides) purchased from a Japanese market. Its structure was spectroscopically identified as 4',5-dihydroxy-3',6,7,8-tetramethoxy flavone (8-methoxycirsilineol, 1). This is the first report describing a strong inhibitor of CYP1A2 and 3A4 isolated from Thymus saturoides.

A powerful inhibitor of TNF-alpha production, vialinin A, was synthesized from sesamol through a series of reactions involving double Suzuki-Miyaura coupling, 2,3-dichloro-5,6-dicyano-1,4-benzoquino (DDQ) mediated de-methoxymethylation and oxidative removal of methylene acetal by lead tetraacetate. The synthetic method also made it possible to prepare a related compound, terrestrin B.

A first total synthesis of vialinin B, a powerful inhibitor (IC(50) 20 pM) of TNF-alpha production, is described. The key reactions include a double Suzuki-Miyaura coupling of electron-rich aryl bromide with a couple of phenylboronic acids, a Cu-mediated Ullmann reaction, and a LHMDS-promoted phenylacetylation. This synthesis proceeded in 11 steps with 18% overall yield from a known sesamol derivative.

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki-Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-alpha production in rat basophilic leukemia (RBL-2H3) cells: IC(50) = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

A chronic toxicity study of kojic acid (KA) was performed using male F344 rats by dietary administration at concentrations of 0 (control), 0.5 and 2.0% for 55 weeks. Body weight gain was suppressed in the 2.0% group. The major hematological findings were decreased red blood cell (RBC) count and hematocrit (Ht) values at both 0.5 and 2.0%. In serum biochemistry, increased aspartate transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP) levels were detected in the 0.5 and 2.0% groups. Histopathologically, single cell necrosis of hepatocytes and proliferation of bile ductules in both treatment groups, and hypertrophy of hepatocytes, granulomas and proliferation of bile ducts in the 2.0% group were increased in incidence, and numbers and areas of glutathione-S-transferase placental-form (GST-P) positive foci were increased in the liver of the 2.0% group. In the thyroids, diffuse follicular cell hyperplasia at 0.5 and 2.0% and focal follicular cell hyperplasia and follicular adenoma at 2.0% were increased. A thyroid follicular carcinoma was also observed at 2.0%. Additionally, increased incidences of hyaline casts and basophilic tubules in the kidneys at 2.0% and microgranulomas containing crystals in the lung in both treatment groups were noted. At 2.0%, hypertrophy of cortical cells in zona fasciculata was also increased in the adrenals. In conclusion, no observed adverse effect level of KA was below 0.5%, which is equivalent to 227 mg/kg body weight/day in male rats.

Increasing attention has been focused on food-drug interactions. We have investigated the inhibitory effect of Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4, the main drug-metabolizing enzymes. Three pulvinic acid derivatives, atromentic acid (1), variegatic acid (2), and xerocomic acid (3), isolated from Boletus calopus and Suillus bovinus, revealed nonspecific inhibitory effects on all four CYPs. Using these compounds, the maximum IC50 values obtained with CYP3A4 in vitro were atromentic acid (1), 65.1+/-3.9 microM; variegatic acid (2), 2.2+/-0.1 microM; and xerocomic acid (3), 2.4+/-0.1 microM. Variegatic acid (2) and xerocomic acid (3) were effective inhibitors, comparable to cimetidine, dicoumarol, erythromycin, safrole, and uniconazole. Variegatic acid (2) and xerocomic acid (3) efficiently reduced ferryl myoglobin in CYPs. Reduction of ferryl heme to ferric heme is likely the mechanism of the nonspecific inhibitory effects of these compounds on CYPs.

Bacillus thuringiensis (Bt) proteins are developed for genetically modified crops and the Bt proteins demonstrate no evidence of toxicity by the oral route in traditional animal models. However, the possible toxicity of Bt proteins under conditions of reduced gastric acid secretion and/or small intestinal damage has not been investigated. In the present study, we therefore evaluated following four F344 rat groups with a purified Bt protein Cry1Ab from B. thuringiensis var. Kurustaki HD-1. Gastrointestinal impairment (GI) alone and GI+Bt protein fed (GI+Bt) groups were given i.p. injections of famotidine to reduce gastric acid secretion twice a day at 30mg/kg body weight in weeks 2 and 4. GI and GI+Bt groups were additionally fed diets containing 80ppm indomethacin for induction of intestinal damage during weeks 1 and 3. Bt alone and GI+Bt groups were also fed diet containing Bt protein Cry1Ab at a concentration of 10ppm in weeks 2 and 4. A no treatment control group was also included. At the end of week 4, all animals were euthanized under ether anesthesia, blood samples were collected for hematology and serum biochemistry and a complete necropsy was performed. No significant changes indicative of toxicity of the Bt protein Cry1Ab used here were noted with any of the parameters investigated. In conclusion, no significant toxicological effects were detected in this subchronic gastrointestinal impairment rat model.

Thelephora vialis is a mushroom that grows in symbiosis with pine trees in Yunnan, China, a place known to have some of the richest and most diverse bioresources in the world. This is one of the most favored edible mushrooms, due to its flavor. Our screening for bioactive compounds from these mushrooms isolated a novel potent antioxidant, vialinin A, together with known compounds, from the dry fruiting bodies of T. vialis. Vialinin A is a terphenyl derivative and was elucidated by spectroscopic and chemical methods. Vialinin A showed anti-allergic activities, inhibition of beta-hexosaminidase, tumor necrosis factor (TNF)-alpha, interleukin 4 and monocyte chemotactic protein 1 release from RBL-2H3 cells, whereas atromentin and an inseparable mixture of ganbajunins D and E showed no such effects. Vialinin A displayed potent inhibition of TNF-alpha production from RBL-2H3 cells (IC50, 0.09+/-0.01 nM), indicating stronger inhibition than tacrolimus for organ transplantation (IC50, 0.25+/-0.03 nM). The potent inhibitory activities of these compounds against TNF-alpha production indicate promising new candidates for anti-allergic agents.

Vialinin A, a powerful inhibitor (IC50 90 pM) of TNF-alpha production, was synthesized from sesamol in 11 steps with 28% overall yield. The key reactions include a double Suzuki coupling of electron-rich aryl triflate with phenylboronic acid and an oxidative deprotection of bis-MOM ether. In addition, the related synthetic studies also suggest the necessity for structural revision of ganbajunin C, a positional isomer of vialinin A.

We previously demonstrated that thyroid capsular inflammation induced by continuous treatment with the antithyroidal agent sulfadimethoxine is associated with development of invasive follicular cell carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The inflammatory changes are characterized by large numbers of macrophages and lymphocytes as well as fibroblasts and we hypothesized that it might be enhanced by interplay between macrophages and T cells. To clarify this hypothesis, a comparative study was conducted between athymic nude (rnu/rnu) rats and euthymic (rnu/+) littermates initiated with DHPN (2800 mg/kg, s.c.) followed by sulfadimethoxine treatment in drinking water (0.1%) for 10 weeks. In rnu/+rats, marked capsular thickening with inflammation was induced along with invasive follicular cell carcinomas (2.8 +/- 1.3/rat). In rnu/rnu rats, limited fibrous capsular thickening was noted with or without minimal inflammatory change, and the multiplicity of invasive carcinomas was significantly lower (1.1 +/- 1.0/rat, P < 0.01). Inducible nitric oxide synthase expression in the inflamed lesions was detected in three of 10 rnu/+rats but in none of the rnu/rnu animals. The results thus suggest that development of invasive carcinomas is enhanced by capsular inflammation mediated by T cells, and inducible nitric oxide synthase induction may play a role in tumor progression.

Arctiin, a plant lignan, is metabolized to hormone-like compounds with weak estrogenic and antioxidative activity in experimental animals and man. To clarify its influence on mammary carcinogenesis, female rats were administrated 7,12-dimethylbenz(a)anthracene (DMBA) once, and when the incidence of palpable mammary tumors reached 50%, subjected to ovariectomy (OVX) and divided into tumor-bearing [DMBA-Tumor (+)] and no-tumor-bearing [DMBA-Tumor (-)] groups, subgroups of each then being fed soybean-free diet containing 0, 40, 200, and 1000 ppm of arctiin for 31 wk. The incidence and multiplicity of palpable tumors in the 200 ppm DMBA-Tumor (+) subgroup from week 12 of arctiin treatment tended to be decreased as compared to the 0 ppm subgroup and at terminal sacrifice, the volume of histopathologically defined mammary tumors was decreased in the 40 ppm DMBA-Tumor (-) subgroup, but again without statistical significance. In conclusion, weak inhibitory effects of arctiin on DMBA-induced mammary tumor development were suggested in OVX rats, but any further assessment is needed to obtain conclusive results.

A novel dibenzofuran compound designated vialinin B was isolated from dry fruiting bodies of an edible mushroom, Thelephora vialis, and potently inhibits TNF-alpha production in RBL-2H3 cells (IC(50)=0.02nM) and is a promising anti-allergic agent.

Effects of intestinal damage on thyroid carcinogenesis due to amitrole (AT) were examined in F344 male rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). In experiment 1, rats were provided with diet containing 0.03% AT for 20 weeks after a single subcutaneous injection of DHPN (2800 mg/kg body weight), and concomitantly received 0.01% indomethacin (IM) in the diet to cause small intestinal damage or 1% dextran sodium sulfate (DSS) in the drinking water for induction of colitis following a schedule of intermittent 1-week administration and 1-week withdrawal for a total of 10 times. Groups without AT- and/or IM or DSS treatment were also included. Histopathological examination revealed significant reduction in the incidence and multiplicity of follicular cell adenomas and adenocarcinomas in the group concomitantly treated with IM, but no change in the DSS group, as compared with the AT alone group. In experiment 2, rats were similarly fed diet containing AT for 3 weeks with concomitant IM or DSS treatment after a DHPN initiation, and serum thyroid stimulating hormone levels were found to be significantly elevated only in the IM case. The increase in thyroid follicular cell proliferation due to AT was also clearly suppressed in the group concomitantly treated with IM. From these findings, IM-induced intestinal damage may inhibit thyroid carcinogeneisis in rats, although contributions of other factors, such as a direct inhibitory effect of IM to thyroid follicular cell proliferation cannot be ruled out.

We have established a medium-term colorectal carcinogenesis rat model initiated with 1,2-dimethylhydrazine (DMH) followed by dextran sodium sulfate (DSS) treatment, featuring induction of neoplastic lesions within 10 weeks. In the present study, we examined its ability to detect modification of colon lesion development with 10- or 20-week experimental periods. F344 male rats were given three subcutaneous injections of DMH (40 mg/kg b.w.) in a week followed by free access to drinking water containing 1% DSS for a week. One week after this regimen, basal diet alone, basal diet containing 0.04% nimesulide or 2% lactoferrin as known inhibitors, 0.3% deoxycholic acid (DCA) as a promoter or 1.5% 1-hydroxyanthraquinone (1-HA) as a carcinogen were supplied. At week 10, the incidence and multiplicity of combined adenomas and adenocarcinomas were significantly (P < 0.05 or 0.01) decreased by nimesulide and lactoferrin, and values for adenomas were significantly (P < 0.01) increased in the 1-HA group. There was no clear change in the DCA group. At week 20, multiplicity and volume of the tumors were significantly (P < 0.01 or 0.05) decreased by nimesulide, but no effect was now evident with lactoferrin. Multiplicity and volume of tumors were significantly (P < 0.01) increased in 1-HA group and a similar tendency was apparent (P = 0.08) with DCA. It is concluded that this system offers a useful tool for detection of colorectal carcinogenesis modifiers within 10-20 weeks, pending further studies for verification employing other model chemicals.

Estrogen exposure during the adult period is widely known to promote tumor development in the female genital system, as well as in the mammary gland in experimental animals, but its carcinogenic potential with exposure at the prepubertal stage, for 6 weeks after birth, is not completely understood. In the present study, we therefore evaluated the modifying effects of prepubertal ethinyl estradiol (EE) treatment on susceptibility to multiple organ carcinogenesis with subsequent carcinogen exposure in F344 rats. Dams during the lactation period and their weaned offspring until postnatal-week 6 were fed diet containing 0, 0.2 or 1.0 ppm EE. The offsprings were then administered 7,12-dimethylbenz[a]anthracene (DMBA, 50mg/kg body weight) by gavage for mammary tumor induction in week 7 and given free access to drinking water containing N-bis (2-hydroxypropyl)nitrosamine (DHPN, 0.2%) for wide spectrum tumor induction in organs such as the thyroid, liver, lung and kidney from weeks 6-14. Male and female offspring were euthanized at weeks 27 and 36, respectively, for histopathological examination. While the incidence and multiplicity of mammary tumors showed a tendency for increase in females of the 0.2 and 1.0 ppm EE groups, this was without statistical significance. Furthermore, prepubertal EE exposure did not affect tumor induction in the thyroid, liver, lung, kidney, esophagus, ovary and lymphoid tissue in either sex. The present results thus indicate a lack of influence of estrogen early in life on carcinogenic susceptibility, although the possible impact on mammary carcinogenesis requires further examination.

We have previously reported that thyroid capsular inflammation induced by sulfadimethoxine (SDM), a goitrogen, might play a role in development of invasive follicular cell adenocarcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The present study was designed to examine the role of cyclooxygenase (COX)-2, widely known to be up-regulated in inflammatory states, during chemically induced rat thyroid carcinogenesis. Male F344 rats received a subcutaneous DHPN (2800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing antithyroidal propylthiouracil (PTU, 0.003%) or SDM (0.1%) for 4 or 10 weeks. Control groups receiving goitrogen alone and no treatment were also included. At week 4, diffuse follicular cell hyperplasia was induced in all PTU- and SDM-treated groups, along with fibrous capsular thickening and capsular thickening with inflammation, respectively. Additionally, multiple focal follicular cell hyperplasias and adenomas were observed in the DHPN + PTU and DHPN + SDM cases. At week 10, adenocarcinomas invasive to the capsule and restricted to the capsular adjacent region, were frequent in the DHPN + SDM group, but not observed in the animals given DHPN + PTU. Western blots and immunohistochemistry revealed constitutive COX-2 expression in non-neoplastic follicular cells of the control and all of the PTU- and SDM-treated rats. However, COX-2 reactivity was significantly reduced or negative in the preneoplastic/neoplastic lesions in the DHPN-treated groups. In fibrous or inflamed thickened capsules, only a few component cells with inflammatory elements were positive for COX-2, and there was no significant difference in this regard between the PTU and SDM treatments. The present results suggest that capsular inflammation could play a role in development of invasive carcinomas, but COX-2 expression does not make a major contribution.

Kojic acid (KA) has been used as a food additive for preventing enzymatic browning of crustaceans and as a cosmetic agent for skin whitening. In the present experiments, effects of KA on the induction of hepatic pre-neoplastic lesions in N-bis(2-hydroxypropyl)nitrosamine-initiated (experiment 1) and non-initiated (experiment 2) models, and its promoting influence in a medium-term liver bioassay (experiment 3) were investigated at dietary doses of up to 2% in male F344 rats. In experiment 1, 2% KA feeding induced significant increases in numbers (22.3 +/- 13.0 vs 8.5 +/- 3.4 in the 0%) and areas (0.37 +/- 0.29 vs 0.05 +/- 0.03 in the 0%) of glutathione-S-transferase P form (GST-P)-positive foci and toxic changes such as vacuolation of hepatocytes and microgranulomas. The development of GST-P-positive foci was pronounced in the animals with hepatocellular toxic changes. In experiment 2, numbers (0.65 +/- 0.57 vs 0.17 +/- 0.28 in the 0%) and areas (0.005 +/- 0.005 vs 0.0007 +/- 0.0012 in the 0%) of GST-P-positive foci and hepatocellular proliferating cell nuclear antigen (PCNA) expression (3.8 +/- 2.3 vs 2.6 +/- 0.7 in the 0%) were significantly increased by the 2% treatment. The PCNA-positive hepatocytes were abundantly localized around the vacuolated and granulomatous legions in both experiments 1 and 2. In experiment 3, significant increases in numbers (16.9 +/- 3.2 vs 8.4 +/- 2.7 in the 0%) and areas (1.62 +/- 0.39 vs 0.77 +/- 0.34 in the 0%) of GST-P-positive foci were again observed with 2% KA. These results demonstrate tumor-promoting and possible hepatocarcinogenic activity of KA at 2%, but the carcinogenic potential is likely to be weak. This study also indicated that enhanced replication of hepatocytes related to toxic changes might be involved as an underlying mechanism.

A 2-stage thyroid follicular carcinogenesis model in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) is widely used to detect modifying effects of chemicals on thyroid carcinogenesis. A number of goitrogens are known to strongly promote carcinogenesis, and the carcinomas often originate adjacent to the thyroid capsule and show invasive growth into the capsule or adjacent tissues. To clarify mechanisms of progression to invasive carcinomas, we sequentially evaluated histopathological and immunohistochemical characteristics of thyroids in male F344 rats treated with sulfadimethoxine (SDM, 0.1% in drinking water) for 0-10 weeks beginning 1 week after DHPN initiation (2800 mg/kg body weight, single s.c. injection). In DHPN-SDM-treated rats, multiple focal hyperplasias and adenomas developed in thyroid follicular parenchyma at weeks 4 to 6. Apart from the proliferative lesions, capsular thickening with inflammatory cell infiltration, mainly consisting of macrophages, and migration of follicular epithelium into the capsule were also observed. Focal hyperplasias/adenomas adjacent to the capsule progressively developed to invasive carcinomas at weeks 6 to 10. In thyroid parenchyma, malignant lesions were seldom observed. With SDM-treatment alone, although no neoplastic lesions were observed, capsular thickening with inflammation and epithelial migration resulted in intracapsular residual follicles. Intracapsular residual follicular cells as well as invasive and intrathyroidal carcinoma cells generally showed increased cell proliferative activity, coincidental with cytoplasmic/nuclear positivity for beta-catenin. These results suggested that beta-catenin activation related to capsular inflammation may play a role in development of invasive carcinomas but is insufficient for tumor formation by itself. Whether this is associated with mutations in the beta-catenin gene remains to be clarified.

To establish a rapid bioassay system with neoplastic end-points for detection of colorectal carcinogenesis modifiers, we evaluated the effects of dextran sodium sulfate (DSS) treatment on the different stages of carcinogenesis in rats initiated with 1,2-dimethylhydrazine (DMH). F344 male rats were given three subcutaneous injections of DMH (40 mg/kg body weight) in a week, and were administered drinking water containing 1.0% DSS ad libitum either during or after the initiation period for a week, or both during and after initiation periods for 2 weeks. At the 10th week of the experiment, although the numbers of aberrant crypt foci were significantly decreased in all groups treated with DSS and given DMH-initiation as compared with DMH alone, dysplastic foci/adenomas/adenocarcinomas were increased. The incidences and multiplicities of these lesions were highest in rats treated with DSS after DMH-initiation period. At the 26th week, the incidences of adenocarcinomas (100 vs. 20% in DMH alone) and their multiplicities (6.6 +/- 0.8/rat vs. 0.2 +/- 0.4/rat in DMH alone) were also highest in this group. These results indicate that short-term DSS-treatment in the post-initiation period significantly accelerates DMH-induced colorectal tumor development in rats, so that this protocol may effective for establishment of a rapid bioassay system with neoplastic end-points.

A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals., ((C)2002 Elsevier Science (USA).)