MER-25 and U-11, 100A, two antiestrogens with tissue-selective and incomplete estrogenic activity, on rat uterus

The effects of the antiestrogenic antifertility agents MER-25 and U-11,100A (nafoxidine), on the uterus of the spayed rat have been studied and compared with those produced by estradiol-17β.In the Allen-Doisy test, all agents were estrogenic. At doses roughly equiactive in this test, the antiestrogens produced marked hypertrophy of the luminal epithelium similar to estradiol-17β but did not increase the mitotic activity in contrast to estradiol-17β. The response to the antiestrogens also appeared to be self-inhibitory with time.The conclusion is made that the complexity of these responses invalidates terms such as estrogenic or antiestrogenic and suggests a need for a more precise terminology.     

The antifertility effectiveness of copper/low-density polyethylene nanocomposite and its influence on the endometrial environment in rats

PURPOSE: The study was conducted to investigate the antifertility effectiveness of copper/low-density polyethylene nanocomposite (nano-Cu/LDPE) and its influence on the endometrial environment in rats. METHODS: One hundred and seventy sexually mature female Sprague-Dawley (SD) rats were randomly divided into five groups: sham-operated control group (SO group, n=10), bulk copper group (Cu group, n=40), LDPE group (n=40), and nano-Cu/LDPE groups I (n=40) and II (n=40). Twenty rats in each group except for the SO group were mated with male rats of proven fertility, from 30 days after insertion, and the antifertility rates (ATs) were observed at Day 11 of pregnancy. The concentrations of prostaglandin E(2) (PGE(2)) and tissue plasminogen activator (tPA) in the endometrium of the remaining rats in each group were measured by using ELISA at the 30th and 60th day after insertion, respectively. RESULTS: ATs in the Cu group and nano-Cu/LDPE groups I and II (100%) were significantly higher than those in the LDPE group (p<.05). Compared with those in the SO group, the concentrations of PGE(2) and tPA in all experimental groups except for PGE(2) levels in the LDPE group were significantly increased at Day 30 after insertion, and these parameters in the nano-Cu/LDPE groups were significantly lower than in the Cu group (p<.05). At Day 60 after insertion, tPA levels were still higher in the Cu and nano-Cu/LDPE groups, but there was no difference in PGE(2) levels in all groups except for the Cu group. CONCLUSION: Nano-Cu/LDPE exhibits satisfactory contraceptive efficacy with less influence on the endometrium PGE(2) and tPA levels.     

The effect of Maillard reaction products on zinc metabolism in the rat

The effect of giving Maillard reaction products (MRP) on zinc metabolism was investigated in the rat. In Expt 1, MRP were prepared by incubating casein with either glucose or lactose under controlled reaction conditions, and were quantified as either 'early' or 'advanced' after estimation of lysine loss and lysine destruction respectively. In Expt 2, the effect of the purified early MRP fructose--lysine (FL) on Zn metabolism was studied. The experimental diets containing 20 mg Zn/kg were given to weanling rats for 21 d. Zn balance was assessed over 9-14 d (Expt 1), or 1-14 d (Expt 2). Femur, liver, kidney and serum Zn concentrations were determined at 21 d. The major effect of the MRP in the casein-sugar mixtures was on urinary Zn excretion. The casein-glucose MRP induced up to a 6-fold increase in the quantity of Zn excreted in the urine. The magnitude of the hyperzincuria increased with the extent of the Maillard reaction. Similar dietary levels of casein-lactose MRP increased urinary Zn loss 2-fold. Free FL had no effect on urinary Zn. Faecal Zn, Zn retention, liver, femur and serum Zn were generally not influenced by giving MRP from casein-sugar mixtures or by giving free FL, although kidney Zn was decreased in rats fed on FL. It was concluded that although urinary Zn excretion can be increased by the presence of MRP in the diet, this is only a minor excretory pathway and would have little influence on overall Zn nutrition in individuals fed on a diet adequate in Zn.     

Effect of estradiol on heme biosynthetic pathway in lead-poisoned rabbits

Objectives To clarify the effect of the female hormone estradiol (Est) on heme biosynthesis in lead-poisoned rabbits, parameters indicating lead exposure, such as free erythrocyte protoporphyrin (FEP) level and δ-aminolevulinic acid dehydratase (ALA-D) activity, were determined. Methods Twenty-six male Japanese white rabbits (body weight (BW), 3kg) were divided into four groups: I (control), II (Est), III (Pb), IV (Est+Pb). About 3 weeks after castration, Est (3 mg/kg of BW) was injected intramuscularly, and 2 weeks thereafter, lead (1.2 mg/kg of BW) was injected intravenously. After the initial injection of each of these substances, the same dose of each of these substances was injected once a week until the 9th week. Results In groups III and IV, FEP level increased and ALA-D activity in the erythrocytes, bone marrow and liver decreased with an increase in lead concentration in blood. FEP level decreased significantly (p<0.01) in the 8th and 10th weeks after Est injection in group IV compared to with that in group III and was not elevated in group II compared with that in group I. ALA-D activity in the erythrocytes, bone marrow and liver increased significantly in group II compared with that in group I, whereas Ht and Hb levels decreased in group II compared with those in group I, and decreased in group IV compared with those in group III. The level of iron in plasma (Fe−P) was within the normal range during experiment. Conclusions In this study, Est did not increase FEP level. From the above results regarding FEP level and ALA-D activity, Est may prevent an increase in FEP level caused by lead. Ht and Hb levels, which are the parameters of anemia, decreased mainly as a result of Est exposure rather than lead exposure.     

The antifertility activity and toxicity of alpha-chlorohydrin derivatives in male rats.

The antifertility activity and toxicity of alpha-chlorohydrin, six derivatives and glycidol were investigated in male rats. At a dose of 5 mg/kg injected intraperitoneally each day for 14 days, only alpha-chlorohydrin produced complete infertility. When rats were injected with the derivatives at a dose equivalent to 5 mg/kg of alpha-chlorohydrin on a molar basis, the phosphorylated and amino acid derivatives produced complete infertility within 7 days; however, there were some signs of toxicity. Compound II also produced complete infertility and appeared less toxic. AY - 22,352, compounds III and IV and glycidol had no antifertility activity, although spermatozoan motility was reduced by injection of glycidol.     

Effects of gossypol on the hepatic drug metabolizing system in rats

Gossypol, a polyphenolic pigment found in cotton plants, has been implicated as an antifertility agent. This pigment has been shown to alter myriad metabolic pathways. The objective of this study was to determine the effect of gossypol acetic acid on the hepatic microsomal drug metabolizing enzyme system in adult female Fisher 344 rats. Subcutaneous administration in both low (15 mg/kg) and high (30 mg/kg) doses of gossypol acetic acid for five consecutive days significantly (P < 0.00001) reduced hepatic aniline hydroxylase activity, as well as cytochrome P-450 and b₅ levels. Thus, compared to the controls, the low doses of gossypol decreased the hepatic aniline hydroxylase activity and cytochrome P-450 and b₅ content by 73, 54, and 43 percent, respectively. The high doses of gossypol decreased the aniline hydroxylase, cytochrome P-450, and b₅ levels by 60, 51, and 46 percent, respectively, as compared to controls. These data indicate that alteration of the hepatic metabolizing system may, in part, be responsible for the secondary toxemic effects seen when gossypol is used to provide antifertility action.     

Sex difference of free erythrocyte protoporphyrin (FEP) level. III. Effect of estradiol on porphyrin metabolism, especially in FEP, in lead poisoned rats]

An experimental study of sex hormone on FEP level in rats was conducted to clarify sex difference of FEP level. A total of 42 male Donryu rats were divided into 7 groups; group I (control 1; olive oil injection without castration), group II (control 2; olive oil alone), group III (EL; estradiol in olive oil, 50 micrograms/kg.BW), group IV (EH; estradiol in olive oil, 250 micrograms/kg.BW), group II' (Pb; Pb 5 mg/kg.BW), group III' (PbEL; Pb 5 mg/kg.BW + estradiol in olive oil, 50 micrograms/kg.BW) and group IV' (PbEH; Pb 5 mg/kg.BW+estradiol in olive oil, 250 micrograms/kg.BW). Excluding group I, all rats of the 6 groups were castrated. After one week from castration, estradiol was subcutaneously injected 4 times a week, and lead was intraperitoneally injected once a week for 6 wk. Estradiol suppressed growth, Ht, Hb value and liver function, but did not show any evident effect on FEP, ALA-U and CP-U. Erythrocyte ALA-D activity in group IV' (Pb+estradiol at high dose) showed a level higher than that in group II' (Pb-treatment alone).     

Effect of sulfasalazine and its analogs on fertility in male rats

Several derivatives of sulfasalazine were tested for their antifertility activity in male rats. The compounds were administered to groups of rats daily by oral gavage for 28 days. Fertility of the rats treated with sulfasalazine or compound CH 74A was reduced, while other compounds had no effect. In a subsequent experiment, therefore, only the active compounds were studied further. Fertility of rats treated with sulfasalazine, compound CH 74A, CH 99A or sulfapyridine was reduced during 40 days of treatment. At the end of treatment, body weights were reduced in higher dose groups of sulfasalazine, CH 74A and sulfapyridine compared to control animals. The weights of the testes, prostate or seminal vesicle were not altered by any of the treatments. On the other hand, weight of the epididymides decreased in all higher dose groups except in CH 99A-treated animals. Sperm motility decreased in all the treated rats except in animals treated with low dose of sulfapyridine, whereas epididymal sperm count decreased in all but CH 99A-treated animals. These results suggest that sulfasalazine and its derivatives bring about their antifertility effects by decreasing sperm motility and/or number of spermatozoa.     

Mode of action of the antifertility sulphonamides: Lack of effects on folate metabolism

The effects of some antifertility sulphonamides on folate metabolism were investigated in the male rat. Subcutaneous injections of sulphanilamide at a dose of 150 mg/kg/day for 6 weeks produced a marked reduction in fertility of the treated animals. This effect was rapidly recovered by one week after drug withdrawal. Similar treatments with trimethoprim (30 mg/kg/day) or pyrimethamine (8 mg/kg/day) had virtually no effect on fertility. The synergistic effect of trimethoprim or pyrimethamine on the antifertility activity of sulphanilamide was not observed when the drugs were administered in combinations. Treatment with sulphapyridine (450 mg/kg/day for 6 weeks) failed to alter the levels of folate in the blood and the reproductive organs except the testes in which accumulation of folic acid occurred. The results suggest that the antifertility activity of sulphanilamide, sulphapyridine and perhaps some other sulphonamides is not associated with the inhibition of folate metabolism.     

Tissue distribution of quercetin in pigs after long-term dietary supplementation

Although the flavonol quercetin is intensively investigated, our knowledge about its bioavailability and possible target organs is far from being complete. The aim of this study was to check the potential of quercetin to accumulate in various tissues after long-term dietary treatment compared with a single treatment with flavonol. Pigs ingested either a single dose of quercetin aglycone (25 mg/kg body weight; Expt. 1) or received the flavonol twice a day at the same dose mixed into their regular meals (i.e 50 mg.kg(-1).d(-1)) for 4 wk (Expt. 2). In both experiments, we took plasma and tissue samples 90 min after the final meal and analyzed them using HPLC. Additionally, the specific activity of the enzyme beta-glucuronidase was measured in selected tissues. Higher flavonol concentrations than in plasma were found in only the liver (Expt. 1) or the intestinal wall and kidneys (Expt. 2). All tissues except blood plasma contained a variable amount of deconjugated quercetin in the range of 30-100% of total flavonols. However, the specific beta-glucuronidase activity was not correlated with the proportions of deconjugated flavonols in the various tissues. Long-term dietary intake of the flavonol did not lead to a greater accumulation in any tissue compared with the single treatment. Flavonol concentrations only exceeded the plasma concentration within organs involved in its metabolism and excretion, including liver, small intestine, and kidneys.     

Effects of metallothionein on mutagenicity and oxidation of quercetin.

The effects of four types of Zn/Cd-metallothioneins on the mutagenicity of quercetin in Salmonella typhimurium TA98 were studied. The four types of Zn/Cd-metallothionein used in this experiment were metallothionein I, metallothionein I/II and metallothionein II from rabbit liver and metallothionein I/II from horse kidney. All four of the metallothioneins enhanced the mutagenicity of quercetin. Metallothionein II from rabbit liver, in which Zn content was the highest of the four metallothioneins, enhanced the mutagenicity of quercetin most effectively. Metallothioneins as well as Zn/Cu-SOD prevented quercetin oxidation under aerobic conditions. The action of these metallothioneins, which enhances the mutagenicity of quercetin, was fairly proportional to the activity which prevents quercetin from being oxidized. Reduced glutathione did not enhance the mutagenicity of quercetin although it did inhibit the quercetin oxidation. These results suggest that metallothioneins have superoxide (O2-) scavenging ability and act as "SOD-like" proteins in vivo. A simple and reliable system to detect biological substances which have antioxidant activity for the superoxide (O2-) was devised.     

Leukotoxin diols from ground corncob bedding disrupt estrous cyclicity in rats and stimulate MCF-7 breast cancer cell proliferation

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (iso-leukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.     

雌激素替代治疗对大鼠动脉粥样硬化的影响

目的:了解早期经皮雌激素替代治疗(estrogen replacement therapy,ERT)对高脂喂养的去势雌性大鼠动脉粥样硬化水平的影响。方法:28只大鼠随机分为4组:A组为假手术对照组,B组为单纯去势组,C组为去势+小剂量ERT组,D组为去势+大剂量ERT组。4组小鼠均给予高脂饮食12周,分别于术前、高脂喂食4周和12周后抽血测定血脂、雌二醇含量及血清SOD活力。结果:12周后B组小鼠血清雌二醇含量明显低于A组(P<0.05),D组明显高于A组(P<0.05),C组与A组间比较差异无统计学意义(P>0.05);12周后B组小鼠血清TC、LDL-C水平显著高于A组(P<0.05);C、D组小鼠的血清TC、LDL-C水平均低于A组(P<0.05),而B组的TG、HDL-C水平均低于其余3组。B组主动脉粥样斑块面积明显大于A、C及D组(P<0.05),C、D两组的斑块面积小于A组(P<0.05);B组血清SOD活力明显下降。结论:早期ERT可改善血脂代谢,抑制血清SOD活性的下降,从而抑制大鼠动脉粥样硬化形成。     

Comparative effects of some steroidal and nonsteroidal antifertility agents in rats and hamsters

The uterotrophic, anti-uterotrophic and contraceptive properties of several antifertility agents vere compared in female rats and hamsters. The compounds fell into two distinct groups on the basis of their biological profile in rats: 1. Estrogenic contraceptives, including estradiol-17β (E₂), 17α-ethynylestradiol (EE), diethylstilbestrol (DES), ORF 3858 and F 6066; 2. Anti-estrogenic contraceptives, including CN55,945-27, U11,555A, Su 13320 and MER-25. These classifications did not hold true for hamsters. E₂ and DES were only 4–5% as active in hamsters as in rats as uterotrophic or contraceptive agents; other estrogens were even less effective. For example, hamsters required 400 to 700 times more ORF 3858 than did rats for uterotrophic and contraceptive activity. Furthermore, enormous doses of all but one of the “anti-estrogenic” compounds failed to antagonize the uterotrophic action of E₂ or to impair fertility in hamsters. The data suggest that fertility may be inhibited in rats by administration of estrogens in highly uterotrophic doses or by treatment with compounds which antagonize the endogenous estrogen required for implantation and maintenance of pregnancy. In contrast, hamsters are less sensitive than rats to the contraceptive effects of estrogens and appear to respond minimally to compounds which are anti-estrogenic in rats.     

Contraceptive and hormonal properties of the stem bark of Dysoxylum binectariferum in rat and docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction

BACKGROUND: This study was aimed to investigate the pregnancy interceptive activity of the stem bark of Dysoxylum binectariferum Hook. f. administered during the pre- and peri-implantation periods and immediately after implantation by oral route in adult female Sprague-Dawley rats. STUDY DESIGN: Ethanolic extract and its fractions were administered to female rats on Days 1-10, Days 1-7, Days 1-5 or Day 1 postcoitum by oral route. At autopsy on Day 12, the number and status of corpora lutea and implantations were recorded. For estrogenic activity, ovariectomized immature rats received the test extract or the vehicle once daily for 3 days and at autopsy on Day 4, uterine weight and status of vaginal opening and extent of vaginal cornification were recorded. For antiestrogenic activity, the extract was administered along with ethinyl estradiol. Docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction, to estrogen receptor (ERalpha) was conducted using AutoDock 3.0.5 on a Linux workstation. RESULTS: The ethanolic extract intercepted pregnancy in rats at a daily dose of 500 mg/kg on Days 1-7 postcoitum. On fractionation, the activity was localized in the chloroform fraction, which inhibited pregnancy in all females at the 35-mg/kg dose on Days 1-7, at the 50-mg/kg dose on Days 1-5 or at the single 300-mg/kg dose on Day 1 postcoitum. Chromatography of this fraction yielded an alkaloid, rohitukine, which prevented pregnancy at the 10-mg/kg dose administered on Days 1-7 but was partially (45%) effective at this dose when administered during the entire preimplantation period and ineffective even at 10 times this dose when administered only on Day 1 postcoitum, except that there was a significant reduction in implantation number in pregnant females. While the active chloroform soluble fraction was devoid of any estrogen agonistic or antagonistic properties, a mild uterotropic effect without induction of premature opening of vagina or cornification of vaginal epithelium was observed in rohitukine at the 10-mg/kg dose. Rohitukine, with an almost similar molecular size (mol. wt. 305) as 17beta-estradiol, fits ideally into the hydrophobic pocket of ER. While it does not appear to simultaneously interact with GLU353, ARG394 and HIS524 as estradiol to elicit frank estrogenic response, different conformations of the ligand or its metabolite(s) might acquire geometry with phenolic groups at C-3', C-5 and C-7 positions disposed in a fashion to interact with active site(s) of ER, which might be responsible for its contraceptive and/or weak uterotropic effects. The absence of a basic side chain directed toward the antiestrogen binding site (ASP351) on the receptor appears to be responsible for the lack of any estrogen antagonistic activity. CONCLUSIONS: Findings demonstrate the antifertility activity of the ethanolic extract of D. binectariferum, its chloroform soluble fraction and rohitukine. Efforts are being made to enhance the anti-implantation activity of rohitukine by structural modifications.     

Antifertility activities of 5-thio-D-glucose in mice and rats

5-Thio-D-glucose given orally to mice at 50 mg/kg/day for up to 49 days inhibited spermatogenesis and fertility, confirming an earlier report. Contrary to the earlier report, the antispermatogenic activity and the drug-induced infertility were only partially reversible as judged by mating trials and testicular morphology. A second experiment, designed to determine if the thiosugar had intrinsic antifertility activity under conditions in which the diabetogenic response was suppressed, showed that the antispermatogenic effect continued to be manifested when insulin (0.5 U/day)-treated rats were given 50 mg/kg/day 5-thio-D-glucose. We conclude that 5-thio-D-glucose is a partially reversible antifertility agent with some apparently intrinsic antispermatogenic activity independent of its effect on serum glucose concentrations.     

Hormonal effects of the 300 μg norethisterone (NET) minipill: 2. Daily gonadotrophin levels in 43 subjects during a pretreatment cycle and during the second month of NET administration

The peripheral plasma levels of immunoreactive follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) were measured daily in 43 normally menstruating women during a pretreatment (control) cycle and during the second month of daily administration of the 300 μg norethisterone (NET) minipill. In addition, the levels of biologically active LH were also determined in 29 of the 43 subjects.

As described in detail in the first paper of these series (1), the 43 women studied exhibited four distinctly different types of ovarian reaction to NET, as indicated by the daily estradiol and progesterone levels. Seven women (16 %) showed neither follicular, nor luteal activity (group A), 10 women (23 %) exhibited a cyclic follicular activity, but no luteal function (group B), 9 women (21 %) had normal follicular function, but insufficient luteal activity (group C), and 17 women (40 %) had estradiol and progesterone levels undistinguishable from those seen in a normal ovulatory cycle (group D).

Administration of the NET minipill did not influence the mean FSH lvel of cycle days 1–6, or those of 3 to 7 days before the LH peak; it slightly decreased the mean luteal phase FSH level in group C, but no in group D, and markedly suppressed the FSH peak value in all groups. There was no difference in this respect between women exhibiting different types of ovarian reaction. Similar to its effect on FSH, the administration of NET did not diminish the mean LH levels of days 1–6, those of 3 to 7 days before the LH peak, or of the luteal phase, but greatly suppressed the LH peak. Again, there was no difference in LH levels during NET administration among women showing different types of ovarian response to the drug. On the other hand, significant differences were found in the LH levels of the pretreatment (control) cycles of the various groups. The mean levels of LH both during days 1–6 and during the luteal phase of the pretreatment cycles were significantly lower in women in whom the minipill subsequently abolished all lutaeal activity (groups A+B) than in women exhibiting different degrees of luteal function (groups C+D). Hence the NET minipill will preferentially inhibit ovulation in women exhibiting relatively low tonic LH-levels in untreated cycles.

The results of the daily LH bioassays were in good agreement with those of the radioimmunoassays.

In the majority of women who exhibited normal (“ovulatory”) estradiol and progesterone profiles during NET administration, the preovulatory FSH, and especially LH peaks were below the lower limit of normal values, and in several instances, normal estradiol and progesterone profiles were found in the virtual absence of any FSH and LH surge.

It is concluded that ovarian suppression by the NET minipill is unrelated to the degree of inhibition of FSH and LH secretion as far as this is reflected by their peripheral levels measured daily.