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.

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.

A human sperm coating antigen isolated from sperm cell membrane.

A human sperm cell membrane antigen that is highly specific to sperm and seminal plasma was isolated from plasma membrane fraction of spermatozoa using rabbit antiserum against human seminal plasma. In addition to the high specificity to sperm and seminal plasma, the isolated antigen showed the following characteristics: (1) It is a glycoprotein of approximately 12,000 daltons that has an affinity to lentil lectin; (2) it is distributed in human milk other than in sperm and seminal plasma, but is not found in any other organs and tissues including testis; (3) seminal plasma contains the largest amount of the antigen activity, 60-fold greater than spermatozoa and 900-fold greater than milk, suggesting that this antigen could be a sperm-coating seminal plasma antigen.

Effect of a soluble factor secreted from cultured human trophoblast cells on in vitro lymphocyte reactions.

The immunoregulatory role of trophoblast cells in cell-mediated immunity was investigated. Trophoblast cells were obtained from 8-10-week human placentae by treatment with collagenase followed by differential centrifugation. The cells were cultured for 48 hr, and the culture supernatant was examined for immunosuppressive activity in vitro. The supernatant when added to cultures of peripheral blood lymphocytes from healthy donors suppressed both their reactivity to different lectins (PHA and PWM) and their activity in one-way mixed lymphocyte reaction. The degree of suppression was dose-dependent. Furthermore, the supernatant was able to reduce the natural killer cell activity against K562 target cells. On the other hand, the supernatant had no inhibitory effect on the effector phase of lymphocyte-mediated cytotoxicity activity against tumor cell lines RPMI 8866 and Daudi. In all cases, the suppression observed was not due to lymphocytotoxicity or tumor cell mortality. The results indicate that trophoblast cells release a soluble suppressive factor that is a potent inhibitor of cell-mediated immunity.

Comparative studies of chromosomal aberration induced by trivalent and pentavalent arsenic.

The comparative cytogenetic effects on the synthesis of DNA, RNA and protein in cultured mammalian cells of trivalent and pentavalent arsenic were investigated. The chromosome-breaking activity in cultured leukocytes was significantly higher for the compounds with trivalent (NaAsO2, AsCl3 and As2O3) than with pentavalent arsenic (Na2HAsO4, H3AsO4 and As2O5). The activity in cultured human skin fibroblasts was similar to that in leukocyte cultures. The colony-forming capacity after exposure to arsenicals indicated that trivalent was more toxic than pentavalent arsenic. In the response of DNA, RNA and protein synthesis, both trivalent and pentavalent arsenic inhibited DNA and protein synthesis in leukocytes.

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions.

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.

Mutagenicity on chlorination of products formed by ozonation of naphthoresorcinol in water.

The mutagenicity of products formed by chlorination after ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated and subsequently chlorinated naphthoresorcinol was directly mutagenic, as was ozonated naphthoresorcinol, in both strains tested. The mutagenic activity at chlorination with 8 equivalents of chlorine per mole of naphthoresorcinol after ozonation was markedly higher than that at only ozonation. Of the identified ozonation products of naphthoresorcinol, muconic acid, after chlorination with 2 or 4 equivalents of chlorine per mole of the compound, induced direct mutagenicity against TA98 and TA100. The chlorination of glyoxal with 0.5 and 1 chlorine equivalents per mole of the compound was shown to produce direct mutagenicity toward TA98. The identification of the chlorination products of these compounds is also discussed.

DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal.

The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 mg/ml glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg/kg body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 mg/kg glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.

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.

Comparative studies of chromosomal aberration and mutagenicity of the trivalent and hexavalent chromium.

The comparative cytogenetic and mutagenic effects between trivalent and hexavalent chromium were investigated. Five chromium compounds, K2Cr2O7 and K2CrO4 containing Cr6+, and Cr(CH3COO)3, Cr(NO3)3 and CrCl3 containing Cr3+, were examined for their ability to induce chromosomal damage in cultures of human leukocytes, for their reactivity with DNA by a rec-assay system and for mutagenicity in the E. coli Hs30R test system. Chromosome-breaking activity was significantly higher for the compounds with hexavalent than trivalent chromium, the efficiency being in the decreasing order K2Cr2OM greater than K2CrO4 greater than Cr(CHCOO)3 greater than Cr(NO3)3, CrCl3. In the rec-assay and mutation assay, hexavalent (K2Cr2O7 and K2CrO4) and trivalent Cr(CH3COO)3) compounds gave positive results, their mutagenic potential being higher in the same order of clastogenic magnitude.

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water.

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.

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.

Studies on selenium-related compounds. V. Cytogenetic effect and reactivity with DNA.

Five selenium compounds, Na2Se04, H2Se04, Na2Se03, H2Se03 and Se02, were tested for their capacity to induce chromosome aberrations in cultured human leukocytes and for their reactivity with DNA by a rec-assay system and inactivation of transforming activity in Bacillus subtilis. Chromosome-breaking activity was significantly higher for the compounds with four-valent than with six-valent selenium, the efficiency being in the decreasing order H2S03 greater than Na2Se03 greater than Se02 greater than H2Se04 greater than Na2Se04. Rec assay using B. subtilis with different recombination capacities suggested that damage to DNA was produced by selenites but not by selenates. The reactivity of selenites with DNA was also indicated by a significant loss of transformation of the tryptophan marker of B. subtilis DNA treated with H2Se03 and Se02.

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.

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.

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.

[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.

[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.