Effect of steroid add-back therapy on the proliferative activity of uterine leiomyoma cells under gonadotropin-releasing hormone agonist therapy.

Short-term treatment with gonadotropin-releasing hormone agonist (GnRHa) is a useful preoperative medical therapy of uterine leiomyomas. However, adverse effects caused by the hypo-estrogen state sometimes appear, suggesting the necessity of add-back therapy. In this study, we investigated effects of three kinds of add-back therapies on the proliferative activity of uterine leiomyoma cells by examining the expression of Ki-67 in leiomyoma cells by immunostaining. Thirty patients who were to undergo hysterectomy or myomectomy were injected with 3.75 mg depot leuprolide acetate every four weeks until the end of the 12th week. Twenty patients underwent add-back therapy from the 5th week to the end of the 12th week, 8 patients receiving 0.625 mg of conjugated equine estrogen (CEE) /day, 6 patients 5.0 mg of medroxyprogesterone acetate (MPA)/day, 6 patients 0.625 mg CEE plus 2.5 mg of MPA /day. The add-back of CEE or CEE plus MPA suppressed decreases in the proliferative activity of leiomyoma cells caused by GnRHa therapy, but that of MPA did not. These results suggest that the add-back therapy with MPA is of use in preventing the adverse effects caused by hypo-estrogen in the preoperative short-term GnRHa therapy.

Contribution of enzymic alpha, gamma-elimination reaction in detoxification pathway of selenomethionine in mouse liver.

The objective of this study was to clarify the detoxification pathways of selenomethionine (SeMet) in mouse liver. It has been postulated that SeMet may be metabolized to selenocysteine (SeCyH) via a pathway similar to methionine (Met). CySeH may be decomposed to H(2)Se, which is consequently methylated to CH(3)SeH, (CH(3))(2)Se, and (CH(3))(3)Se(+). In this study, we estimated that the median lethal single oral dose (LD(50)) was 67.0 mg/kg. We also found that (CH(3))(3)Se(+) was quickly produced in mouse liver after single oral administration of SeMet. This result suggested the existence of a quick alpha,gamma-elimination pathway. We measured the amounts of alpha-ketobutyrate, NH(3), and CH(3)SeH produced by enzymic alpha,gamma-elimination reaction of SeMet in the liver of periodate-oxidized adenosine (PAD) or D,L-propargylglycine (PPG)-treated mice in order to verify the existence of alpha,gamma-elimination enzyme. PAD is an inhibitor of S-adenosylhomocysteinase (EC 3.3.1.1), which is necessary for conversion of SeMet to SeCyH. PPG is an effective inhibitor of the pyridoxal 5'-phosphate (PLP)-containing enzyme bacterial L-methionine gamma-lyase (EC 4.4.1.11) contributing to the alpha,gamma-elimination reaction of SeMet and cystathionine gamma-lyase (EC 4.4.1.1) relating to conversion of SeMet to SeCyH. When SeMet was incubated with the S9 fraction from liver of PAD-treated mice, the formation of alpha-ketobutyrate was much the same as that from nontreated mouse liver. However, the amount of alpha-ketobutyrate formed significantly decreased in the reaction of SeMet with S9 fraction from the liver of PPG-treated mice. In an in vivo experiment using mice treated with PAD before a toxic dosage of SeMet, the amount of SeMet in the liver decreased and the amount of acid-volatile Se derived from CH(3)SeH increased gradually. This phenomenon was not observed in the PPG-pretreated group. Furthermore, the protein fraction that had the alpha,gamma-elimination enzyme activity was found in mouse liver cytosol by gel chromatographic technique. The results of this study indicated that SeMet was directly metabolized to CH(3)SeH by an alpha,gamma-elimination enzyme analogous to bacterial L-methionine gamma-lyase, in addition to the generally acceptable pathway via SeCyH.

The variation on the mutagenicity of CNP during anaerobic biodegradation.

The mutagenicity of water, including herbicide CNP, and its time-variation during anaerobic biodegradation were studied through Ames assay using strains with or without. S9 mix: TA98, TA 100, YG1021, YG1024, YG1026, and YG1029. The bacteria, for the anaerobic biodegradation, was obtained from a paddy field, and preincubated for a month. The CNP was decomposed in an anaerobic culture inoculated with the bacteria, and finally yielded CNP-amino as one of the CNP metabolites. About 16% of the initial CNP was transformed into CNP-amino by the 14th day. The mutagenicities to TA98. YG1024, and YG1029 strains with S9 mix increased with cultivating time, the latter two showed the strongest sensitivity to CNP-amino. The contribution of CNP to the mutagenicity decreased as the chemical decomposed, while the contribution of CNP-amino increased. However, the increased mutagenicity was not limited to the contribution of CNP-amino. but also to the contribution of other metabolites. The contributions of other CNP metabolites were 67% of total mutagenicity to the TA98 strain and 30% to the YG1029 strain. These unknown mutagenic metabolites were the indirect frameshift mutagens which did not have nitro- and amino-substituents, and the indirect base-pair mutagens which might possibly have some amino-substituents.

Degradation of malathion and phenthoate by glutathione reductase in wheat germ.

Residual malathion in wheat was estimated at a lower value when analysis was performed by extraction with acetone after addition of water to swell the wheat, according to the Japanese Bulletin Method. The supernatant of the wheat homogenate showed degradation not only of malathion but also of phenthoate. Malathion and phenthoate were not degraded by the boiled supernatant of the wheat homogenate. It was presumed for this reason that glutathione reductase (GR; EC 1.6. 4.2) in the wheat degraded malathion. The following results were obtained: (1) GR originating in wheat could degrade malathion and phenthoate. (2) The degradation of malathion by the GR was inhibited by excessive GSSG. (3) There was a high correlation between GR activity and malathion degradation activity of the supernatant of wheat homogenates. It is likely that GR acted on the specific structure of malathion and phenthoate, the S=P-S bond, and the blanch structure bonding with the sulfur atom. Following the above, extraction with acetone after addition of water (the Japanese Bulletin Method) should be replaced by extraction with pure organic solvent and without addition of water for swelling.

[Evaluation of endocrine disruptors in environmental water using yeast two-hybrid system].

Estrogenicity of concentrates from waters of lake, river, tap water and effluent of sewage treatment plant by XAD-2 resin column concentration method was detected by the yeast two-hybrid system. Estrogenicity was detected in all environmental waters. From dose-response curve on estrogenic activity of concentrates from the Yodo river water by the two-hybrid system with and without S9mix, 17 beta-estradiol equivalent values in the river water were very similar to analytical values of 17 beta-estradiol reported by the Ministry of Constraction's survey(1999). Estrogenic activities of these concentrates were enhanced by metabolic activation. On the other hand, the tests on effect of these concentrates against estrogenic activity of 17 beta-estradiol (6 x 10(-10) M) revealed that the river water may contain not only inhibitors to estrogenicity but also precursors of estrogenic substances formed by metabolic activation.

Oxidative cell damage in Kat-sod assay of oxyhalides as inorganic disinfection by-products and their occurrence by ozonation.

Nine oxyhalides as possible inorganic disinfection by-products were tested for oxidative cell damage by Kat-sod assay with E. coli mutant strains deficient in the active oxygen-scavenging enzymes. Chlorine dioxide, chlorite, and iodate were highly cytotoxic, whereas in the presence of cysteine, bromate (BrO3-) and metaperiodate (IO4-) showed more growth inhibition toward the superoxide dismutase-deficient strains than the wild strain. BrO3- also showed oxidative mutagenicity with cysteine or glutathione ethyl ester in S. typhimurium TA 100. To identify oxyhalides formed by ozonation of raw water containing sea water, the occurrence of ozonation by-products of bromide and iodide was investigated. The results indicate that BrO3- is toxicologically one of the most remarkable oxyhalides detectable in drinking water because IO4- was not detected in the ozonated solution of iodide, and the ozonation condition to lower BrO3- is to keep it neutral in the presence of ammonium ion.

The gonadotropin-releasing hormone agonist leuprolide acetate induces apoptosis and suppresses cell proliferative activity in rectovaginal endometriosis.

A gonadotropin-releasing hormone agonist, leuprolide acetate, was administered every 4 weeks for treatment of rectovaginal endometriosis. Degrees of apoptosis (percentage of in situ deoxyribonucleic acid 3'-end-labeled cells) and cell proliferative activity (percentage of cells with immunostaining for proliferating cell protein Ki-67) were examined in endometriotic glands of biopsy specimens taken before and during gonadotropin-releasing hormone agonist therapy. Gonadotropin-releasing hormone agonist induced apoptosis and suppressed cell proliferative activity in endometriotic glands.

Suppression of cell proliferation and induction of apoptosis in uterine leiomyoma by gonadotropin-releasing hormone agonist (leuprolide acetate).

Cell proliferation and apoptosis in uterine leiomyoma were investigated during therapy with GnRH agonist (GnRHa). Patients with uterine leiomyomas were injected with 3.75 mg GnRHa (depot leuprolide acetate) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of GnRHa therapy. Tissue sections of leiomyomas from these patients and from control patients (control patients received no GnRHa therapy) were stained with the Ki-67 antibody or by an in situ DNA 3'-end labeling method, and numbers of Ki-67 immunostained cells and DNA 3'-end-labeled cells per cm2 were examined as indices of cell proliferation and apoptosis, respectively. The number of Ki-67 immunostained cells/cm2 in leiomyomas at the 2nd week of the GnRHa therapy was comparable with that of control patients. However, it decreased to a level less than one forth that of control patients at the 4th week, and it remained at similar low levels at the 8th, 12th, and 16th week. The number of DNA 3'-end-labeled cells/cm2 in leiomyomas of control patients and in leiomyomas at the 2nd, 8th, 12th, and 16th weeks of GnRHa therapy were at low levels but, at the 4th week, was at an extremely high level (about 5 times more than that of control patients). The present results indicate that GnRHa therapy suppresses cell proliferation and causes a transient increase in apoptosis in uterine leiomyomas.

[Selenium methylation and toxicity mechanism of selenocystine].

Selenium is an essential trace element and a toxicant for animals. Selenocystine, a selenium-containing amino acid, is one of the chemical forms in which selenium exists in food. This review summarized recent studies on the toxicity mechanism of selenocystine in experimental animals. Hepatotoxicity is caused by repeated oral administration of selenocystine. Selenocystine is metabolized by reduced glutathione and/or glutathione reductase to hydrogen selenide via selenocysteine-glutathione selenenyl sulfide. The hydrogen selenide is a key intermediate in the selenium methylation metabolism of inorganic and organic selenium compounds. Accumulation of the hydrogen selenide resulting from inhibition of the selenium methylation metabolism, detoxification metabolic pathway of selenium, is found in animals following repeated administration of a toxic dose of selenocystine. The excess of the hydrogen selenide produced by inhibition of the selenium methylation metabolism contributes to the hepatotoxicity caused by selenocystine.

Disinfection by-products in the chlorination of organic nitrogen compounds: by-products from kynurenine.

Volatile by-products in the chlorination of 3 humic acids as naturally-occurring substances and 37 nitrogen compounds normally found in excrement were analyzed, and as result kynurenine, a urinary metabolite of tryptophan was found a suitable model compound for dichloroacetonitrile-forming precursors. Possible pathways for the formation of chlorination by-products from kynurenine were also proposed by identification and kinetic properties of by-products decomposed further from each product.

Mechanisms of selenium methylation and toxicity in mice treated with selenocystine.

Mechanisms of selenium methylation and toxicity were investigated in the liver of ICR male mice treated with selenocystine. To elucidate the selenium methylation mechanism, animals received a single oral administration of selenocystine (Se-Cys; 5, 10, 20, 30, 40, or 50 mg/kg). In the liver, both accumulation of total selenium and production of trimethylselenonium (TMSe) as the end-product of methylation were increased by the dose of Se-Cys. A negative correlation was found between production of TMSe and level of S-adenosylmethionine (SAM) as methyl donor. The relationship between Se-Cys toxicity and selenium methylation was determined by giving mice repeated oral administration of Se-Cys (10 or 20 mg/kg) for 10 days. The animals exposed only to the high dose showed a significant rise of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma. Urinary total selenium increased with Se-Cys dose. TMSe content in urine represented 85% of total selenium at the low dose and 25% at the high dose. The potential of Se-methylation and activity of methionine adenosyltransferase, the enzyme responsible for SAM synthesis, and the level of SAM in the liver were determined. The high dose resulted in inactivation of Se-methylation and decrease in SAM level due to the inhibition of methionine adenosyltransferase activity. To learn whether hepatic toxicity is induced by depressing selenium methylation ability, mice were injected intraperitoneally with periodate-oxidized adenosine (100 mumol/kg), a known potent inhibitor of the SAM-dependent methyltransferase, at 30 min before oral treatment of Se-Cys (10, 20, of 50 mg/kg). Liver toxicity induced by selenocystine was enhanced by inhibition of selenium methylation. These results suggest that TMSe was produced by SAM-dependent methyltransferases, which are identical with those involved in the methylation of inorganic selenium compounds such as selenite, in the liver of mice orally administered Se-Cys. Depression of selenium methylation ability resulting from inactivation of methionine adenosyltransferase and Se-methylation via enzymic reaction was also found in mice following repeated oral administration of a toxic dose of Se-Cys. The excess selenides accumulating during the depression of selenium methylation ability may be involved in the liver toxicity caused by Se-Cys.

Identification and metabolism of selenocysteine-glutathione selenenyl sulfide (CySeSG) in small intestine of mice orally exposed to selenocystine.

This investigation was carried out to elucidate the chemical form of selenium-containing metabolite in small intestine of ICR male mice orally administered selenocystine (CySeSeCy). The metabolite in intestinal cytosol of mice treated with CySeSeCy (50 mg/kg) was identified as selenocysteine-glutathione selenenyl sulfide (CySeSG) by high performance liquid chromatography using a gel filtration and reversed phase column. Hydrogen selenide formation was caused as a result of the anaerobic reaction between the CySeSG and liver cytosol containing selenocysteine beta-lyase, which specifically acts on selenocysteine (CySeH). Effects of GSH or glutathione reductase on hydrogen selenide formation from CyseSG reacted with the liver cytosol were examined. The CySeSG was nonenzymatically reduced to CySeH by excess GSH in the liver cytosol. It was also recognized that CySeSG was enzymatically reduced to CySeH by glutathione reductase in the presence of NADPH. These results indicate that the chemical form of this metabolite is CySeSG, which has a molecular weight of 473, the CySeSG is then reduced by excess GSH and/or glutathione reductase yielding CySeH, which is decomposed by selenocysteine beta-lyase to hydrogen selenide. CySeSG may be a stable precursor of hydrogen selenide in animals.

Danazol concentrations in ovary, uterus, and serum and their effect on the hypothalamic-pituitary-ovarian axis during vaginal administration of a danazol suppository.

OBJECTIVE: To determine danazol concentrations in the ovary, uterus, and serum during daily vaginal administration of a danazol suppository and to examine its effect on the hypothalamic-pituitary-ovarian axis. DESIGN: Sampling of tissues after vaginal or oral administration of danazol and sampling of blood during control and danazol-administration menstrual cycles. SETTING: Outpatient volunteers and inpatients at a public hospital. PARTICIPANTS: Thirty patients who were to undergo hysterectomy and oophorectomy because of uterine leiomyoma and eight regularly menstruating volunteers. INTERVENTIONS: Danazol was administered as a vaginal suppository (100 mg) or orally (400 mg). MAIN OUTCOME MEASURE: Danazol concentrations in the ovary, uterus, and serum, and serum E2 and P levels. RESULTS: Danazol concentrations in the ovary and uterus after daily vaginal administration of a suppository containing 100 mg danazol were comparable to those after daily oral administration of 400 mg danazol, but the serum danazol concentration was much lower. Menstrual cycle patterns of serum E2 and P levels were normal during daily vaginal administration of a danazol suppository. CONCLUSION: Daily administration of a suppository containing 100 mg danazol produces high ovarian and uterine concentrations but low serum concentrations, and no effect was detected on the hypothalamic-pituitary-ovarian axis.

Chemical form of selenium-containing metabolite in small intestine and liver of mice following orally administered selenocystine.

The chemical form of a selenium-containing metabolite in the small intestine following a single oral administration of selenocystine was investigated with ICR male mice. Selenium content in the small intestine of animals treated with 50 mg/kg selenocystine significantly increased 15 min, 1 h and 6 h after treatment. In contrast, selenocystine significantly depressed the intestinal reduced glutathione (GSH) level at 1 h after administration. A significant negative correlation between the selenium level and the level of GSH in the small intestine was observed (r = -0.83, p < 0.001). Analysis of the intestinal metabolite of selenocystine showed that selenium-containing metabolites elute in two fractions from a Sephadex G-25 column: the low-molecular fraction (peak I) contained the selenocystine, while the high-molecular fraction (peak II) contained selenocysteine-containing metabolite. An in vitro experiment was performed to gain insight into the mechanism for selenocysteine-containing metabolite production in the intestinal cytosol. When selenocystine or selenocysteine reacted with excess GSH in the presence of intestinal homogenate, the peak II fraction which involved the selenocysteine-containing metabolite was recognized in the Sephadex G-25 chromatogram. From an examination of the distribution of the selenocysteine-containing metabolite, it was recognized that this metabolite exists in plasma and liver cytosol of mice after oral administration of selenocystine. These results suggested that the mice treated with selenocystine produce selenocysteine-containing metabolite by reaction of selenocystine with excess GSH in the small intestine, and the metabolite is then transported to the liver through blood plasma.

Distribution and chemical form of selenium in mice after administration of selenocystine.

To elucidate the relationship between chemical forms of selenium in tissues and subacute liver damage induced by selenocystine (T. Hasegawa et al., Arch. Toxicol., 68, 91 (1994)), the distribution and chemical form of selenium were investigated in ICR male mice treated with the chemical orally (50 mg/kg) and intravenously (5 mg/kg). The time-distribution of selenium in plasma, erythrocytes and liver after separate administration varied. However, Sephadex G-150 chromatograms of plasma, and stroma-free hemolysate from mice treated orally or intravenously with selenocystine, revealed that selenium exists mainly in the albumin and hemoglobin fractions, respectively, and is neither route- or time-dependent. Sephadex G-150 chromatograms of liver cytosol of the animals 1 h after oral administration or 1 and 6 h after intravenous administration showed two selenium-containing fractions, void volume and a low-molecular fraction (Kav = 0.85); 6 h after oral treatment, however, animals had an additional high-molecular fraction (Kav = 0.45). Levels of acid-volatile selenium and dialyzable selenium in the fraction with a Kav value of 0.45 were similar, being 31.2% and 30.3%, respectively. No acid-volatile selenium was recognized in the non-dialyzable high-molecular fraction. The present study demonstrated that when selenocystine is administered orally to mice, the selenium which produces acid-volatile selenium by acidification may bind to protein sulfhydryl groups in the liver cytosol; this was not seen in the case of intravenous administration.

Toxicity and chemical form of selenium in the liver of mice orally administered selenocystine for 90 days.

The subacute oral toxicity of selenocystine and chemical form of selenium in the liver following exposure to this compound were assessed in ICR male mice. Animals were dosed 6 days/week for 30, 60 or 90 days with 0, 5, 10 or 15 mg/kg per day. Body weight gain decreased with dosage. The activities of aspartate aminotransferase and alanine aminotransferase in plasma were significantly elevated at the highest dose level after 60 days and at the two higher dose levels after 90 days of exposure. However, the level of selenium content in the liver was the same at the two higher dosages at both 60 and 90 days of exposure. The subcellular distribution of selenium in the liver from mice treated with selenocystine showed that the major part of the total selenium content, 68.3-72.1%, existed in the cytosolic fraction. Sephadex G-150 chromatograms of liver cytosol of the animals administered selenocystine revealed three selenium-containing fractions which involve glutathione peroxidase (molecular weight 80,000) high molecular (molecular weight 55,000-60,000) and low molecular (molecular weight < 10,000) substances. Selenium content and acid-volatile selenium content in the high molecular weight fraction increased with exposure time to selenocystine. Thus, in a subacute toxicity study selenocystine given for 90 days caused hepatic damage in mice, depending on the acid-volatile selenium content in the liver cytosol.

Identification of polycyclic aromatic hydrocarbons in mutagenic adsorbates to a copper-phthalocyanine derivative recovered from municipal river water.

A study was made to identify polycyclic aromatic hydrocarbons (PAHs) in the mutagenic adsorbate to blue cotton recovered from the water of the Katsura River which is a tributary of the Yodo River, a typical municipal river. As blue cotton bears a covalently bound copper-phthalocyanine derivative which can adsorb PAHs over 3 rings, PAHs in the adsorbate were separated into 4 fractions (I-IV) by Sephadex LH-20 gel chromatography. Fractions III and IV showed high direct and indirect frameshift mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98, especially in YG1024 with S9 mix, whereas these fractions showed less mutagenicity in TA98NR or TA98/1,8-DNP6. These results suggest that mutagenic nitroarenes and aminoarenes are present in both fractions. The retention times of some peaks separated from both fractions using high performance liquid chromatography (HPLC) with a fluorescence detector were identical with those of authentic PAHs. Gas chromatography-mass spectrometry of some HPLC fractions demonstrated that anthraquinone, azulene derivative, quinoline derivative, chrysene and benzo[b]fluoranthene are probably contained in these fractions.

Improvement of long-term prognosis in patients with ovarian cancers by adjuvant sizofiran immunotherapy: a prospective randomized controlled study.

The effect of immunotherapy using sizofiran (SPG) on the prognosis of patients with ovarian cancers was prospectively studied in a total of 68 patients, who were randomly assigned to either a cisplatin, adriamycin and cyclophosphamide (PAC) therapy group or a PAC plus SPG combination therapy group. The survival rate was significantly higher in patients with stage Ic, II or III cancers treated with the PAC plus SPG combination, compared with the patients treated with PAC alone. In the SPG-receiving patients with stage Ic or more advanced cancers who were treated with four cycles or more of PAC, the outcome was improved (Cox-Mantel, p = 0.074; generalized Kruskal-Wallis, p = 0.032). Similar improvement was also observed in the patients with non-serous adenocarcinomas (Cox-Mantel, p-0.076; generalized Kruskal-Wallis, p = 0.045). No side effects attributable to SPG were recorded. The present results suggest that the use of SPG in combination with long-term chemotherapy improves the postoperative prognosis in ovarian cancer patients.

[The equal employment opportunity that began at the pharmacy division of the Osaka Imperial University Hospital from the pre-war era of Showa].

In 1940 when sexual discrimination prevailed in Japan because of the world war, equal sexual employment was carried out at the pharmacy division of the Osaka University through the effort of Prof. Satani (Dean), Dr. Kaneko (Assistant Superintendent of the Kobe Womens' Pharmaceutical College) and Dr. Okazaki (of the Teikoku Womens' Pharmaceutical College) and was put into practice by the Ministry of Health and Welfare by the request of the Superintendent of the Osaka University Hospital. Finally, in 1985 equal employment opportunity was brought into Japan by the instruction of GHQ.

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen.

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.