Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole in the rat (III): Metabolites in the urine and feces

The urinary and fecal metabolites of orally administered 2-tert-butyl-4-hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA) in rats were identified. Samples of 2-day pooled urine and feces of rats given a single intragastric dose of 1 g/kg body wt of tert[butyl-14C]3-BHA (*Bu-3-BHA). tert[butyl-14C]2-BHA (*Bu-2-BHA), [methyl-14C]3-BHA (*Me-3-BHA) or [methyl-14C]-2-BHA (*Me-2-BHA) were analyzed by comparing thin-layer chromatography (TLC) retentions with authentic standards. Conjugated metabolites were identified after enzymatic hydrolysis. Proton magnetic resonance spectroscopy and electron impact mass spectrometry were used for confirmation of the authentic standards. In rats given 3-BHA, a major metabolite in the urine was 3-BHA-glucuronide with a smaller amount of tert-butylhydroquinone (TBHQ)-sulfate, while unchanged 3-BHA and 3-BHA-glucuronide were detected in the feces. In rats given 2-BHA, the main metabolites were the sulfate conjugates of 2-BHA, 4-tert-butyl-5-methoxy-1,2-benzoquinone (2-TBOQ) and the glucuronide of 2-BHA in the urine, while unchanged 2-BHA was found in the feces.     

Studies on a cell sampling method in mass screening for uterine cervical cancer--comparison of data obtained with a cotton tipped applicator, wooden spatula and cotton tipped applicator plus colposcopy

In mass screening tests for uterine cancer carried out Shimane Prefecture, we used three different scraping methods. This summary is a report of a comparison of the findings with each method. Results are as follows: From 1973-1982, 214, 798 Japanese women were examined in mass screening tests. Among the 42, 443 examined using a cotton tipped applicator, there were 15 (0.035%) with mild dysplasia, 8 (0.019%) with severe dysplasia, 6 (0.014%) with carcinoma in situ and 8 (0.019%) with invasive carcinoma. In 38,106 women examined using the spatula, 25 (0.066%) had a mild dysplasia, 10 (0.026%) a severe dysplasia, 18 (0.047%) a carcinoma in situ and 26 (0.068%) an invasive carcinoma. In 134,249 examined using a cotton tipped applicator plus colposcopy, 283 (0.211%) had a mild dysplasia 108 (0.080%) a severe dysplasia, 57 (0.042%) a carcinoma in situ and 78 (0.058%) an invasive carcinoma. Thus, the use of a cotton tipped applicator plus colposcopy leads to higher rates of detection of mild and severe dysplasia. In the group with a negative Papanicolaou smear and for whom the cotton tipped applicator plus colposcopy was used, there were 214 with a mild dysplasia, 65 with a severe dysplasia, 11 with a carcinoma in situ and 7 with an invasive carcinoma. The combination of cotton tipped applicator and colposcopy proved to be the most effective for mass screenings for uterine cancer, as the detection rates for severe dysplasia and carcinoma in situ were higher and there were fewer incidences of false negative results.     

Cyclic mechanical stretch augments hyaluronan production in cultured human uterine cervical fibroblast cells

Hyaluronan (HA) a glycosaminoglycan with high affinity for water molecules stimulates local inflammatory reactions. Parturition causes a dramatic increase in the amount of HA fragments in the uterine cervix, thereby contributing to a rapid softening as well as opening of the cervical canal, i.e. cervical ripening. The aim of this study was to investigate the possible involvement of cyclic distension caused by labour in the augmentation of HA production during cervical ripening. Immunohistochemistry and/or RT-PCR detected hyaluronan synthase (HAS)1, 2 and 3 in samples of human cervical tissue obtained from pregnant women. Labour-like cyclic mechanical stretch for 24, 36 and 48 h significantly enhanced the secretion of HA, from cultured human uterine cervical fibroblast (CxF) cells, 128.7, 151.4 and 173.2%, respectively, concomitant with a significant augmentation of HAS1 (36, 48 h), HAS2 (24, 36 and 48 h) and HAS3 (48 h) mRNA expression. Cyclic mechanical stretch for 12, 36 and 48 h increased molecular size of the HA secreted from CxF cells. In conclusion, cyclic mechanical stretch of the uterine cervix caused by the presenting part of the fetus in labour may contribute to the increase in the secretion of HA during the process of cervical ripening.     

The medium-term results of treatment with hydroxyapatite implants

Although the short-term results of implants with synthetic hydroxyapatite (HA), a bioactive material, have been favorable, few reports have been published concerning medium-term outcomes for this therapy. The authors recently analyzed data supplied by 37 medical facilities nationwide concerning the outcomes of synthetic HA implants [Bonfil (BF); Mitsubishi Materials Corporation, Tokyo] in 138 patients followed up for at least 5 years after treatment. When the data were analyzed, the patients were divided into two groups: the disease site-filling group (62 cases where bone defects created by disease were filled with synthetic HA) and the donor site-filling group (76 cases where bone defects created by bone donation were filled with synthetic HA). In the disease site-filling group, synthetic HA was used in combination with autologous bone or autogenous bone marrow donated from the same patient in 77% of cases. In the donor site-filling group, only synthetic HA was used in most cases. The average follow-up period after implantation was 7.9 years in the disease site-filling group and 9.1 years in the donor site-filling group. Therapy was rated radiographically as "very effective" or "effective" in 81% of patients in the disease site-filling group and in 89% of patients in the donor site-filling group. The therapy was not rated as "ineffective" in any case from either group. These results suggest that synthetic HA may serve as a very useful substitute for cancellous bone if used carefully, with its initial strength taken into account.     

Hyperplastic gastric tumors with spasmolytic polypeptide-expressing metaplasia caused by tumor necrosis factor-alpha-dependent inflammation in cyclooxygenase-2/microsomal prostaglandin E synthase-1 transgenic mice

We showed recently that Helicobacter infection induces expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in the mouse stomach, and that transgenic mice expressing both cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (K19-C2mE mice) develop hyperplastic gastric tumors with inflammatory histopathology. To investigate possible roles of proinflammatory cytokines and acquired immunity in the gastric hyperplasia of K19-C2mE mice, we introduced knockout mutations for tumor necrosis factor-alpha (TNF-alpha; Tnf), interleukin-1 receptor-alpha chain (Il1r1), and Rag2 genes, respectively. Among the compound mutants, only the Tnf (-/-) K19-C2mE mice showed significant suppression of hyperplastic tumors with reduced cell proliferation. In contrast, tumorigenesis remained unaffected in either compound mutants of K19-C2mE containing Il1r1 or Rag2 mutation, indicating that neither interleukin-1beta signaling nor T cell/B cell response was required for the development of hyperplastic tumors. Importantly, spasmolytic polypeptide/trefoil factor 2-expressing metaplasia (SPEM) in the K19-C2mE stomach was also suppressed in the Tnf (-/-) K19-C2mE mice, indicating that TNF-alpha-dependent inflammation is responsible for SPEM development. Because gastric metaplasia to the SPEM lineage is considered as a preneoplastic lesion of gastric cancer, it is possible that inhibition of TNF-alpha-dependent inflammation, together with eradication of Helicobacter, can be an effective prevention strategy for gastric cancer.     

Exploring the chemistry of uncultivated bacterial symbionts: antitumor polyketides of the pederin family

Symbiotic bacteria have long been proposed as being responsible for the production of numerous natural products isolated from invertebrate animals. However, systematic studies of invertebrate-symbiont associations are usually associated with serious technical challenges, such as the general resistance of symbionts to culturing attempts and the complexity of many microbial consortia. Herein an overview is provided on the culture-independent, metagenomic strategies recently employed by our group to contribute to a better understanding of natural product symbiosis. Using terrestrial Paederus spp. beetles and the marine sponge Theonella swinhoei as model animals, the putative genes responsible for the production of pederin-type antitumor polyketides have been isolated. In Paederus fuscipes, which uses pederin for chemical defense, these genes belong to an as-yet unculturable symbiont closely related to Pseudomonas aeruginosa. To study the extremely complex association of T. swinhoei and its multispecies bacterial consortium, we used a phylogenetic approach that allowed the isolation of onnamide/theopederin polyketide synthase genes from an uncultured sponge symbiont. Analysis of the biosynthesis genes provided unexpected insights into a possible evolution of pederin-type pathways. Besides revealing new facets of invertebrate chemical ecology, these first gene clusters from uncultivated symbiotic producers suggest possible biotechnological strategies to solve the supply problem associated with the development of most marine drug candidates.