Correlation of aromatic hydroxylation of 11 beta-substituted estrogens with morphological transformation in vitro but not with in vivo tumor induction by these hormones.

In estrogen-induced cancer, catechol formation from administered steroids has been postulated to be a necessary event for estrogen activation and subsequent damage to cellular macromolecules. In the present study, this hypothesis has been tested using two homologous series of structurally related estrogens: estradiol, 11 beta-methylestradiol, 11 beta-ethylestradiol, 11 beta-methyl-17 alpha-ethinylestradiol, 11 beta-ethyl-17 alpha-ethinylestradiol, 11 beta-methoxy-17 alpha-ethinylestradiol, and 17 alpha-ethinylestradiol. In the Syrian hamster renal carcinoma model, only 11 beta-methylestradiol and 17 alpha-ethinylestradiol were weak carcinogens (2 of 20 and 2 of 24 hamsters with tumors, respectively). The other estrogens tested induced renal carcinoma within 6 to 9 months with an incidence in the 80-100% range. The tumor incidence in vivo did not correlate with the rates of catechol formation by hamster kidney microsomes in vitro. Compared to estradiol (relative rate, 100), catechol formation by the substituted estrogens was significantly lower, ranging from 48 (11 beta-methylestradiol) to 2 (11 beta-methoxy-17 alpha-ethinylestradiol). Kidney DNA of hamsters treated with the four 17 alpha-ethinyl estrogens, when analyzed by 32P postlabeling assay, contained the same set of covalently modified nucleotides the formation of which had previously been found to precede estrogen-induced renal carcinogenesis in vivo. In contrast, relative rates of catechol estrogen formation by BALB/c 3T3 microsomes correlated with induction of morphological transformation of BALB/c 3T3 cells and decreased in the following order: 11 beta-methylestradiol greater than 17 alpha-ethinylestradiol greater than or equal to estradiol greater than 11 beta-ethylestradiol greater than 11 beta-methoxy-17 alpha-ethinylestradiol. The hormonal potencies of several estrogen derivatives studied by various assays did not correlate with in vivo carcinogenic or in vitro cell-transforming activities. It is concluded from these experiments that in cell culture catechol formation and morphological transformation are directly related. In vivo, aromatic hydroxylation of administered estrogens did not correlate with the incidence of estrogen-induced renal carcinoma in Syrian hamsters.

The effect of a copper intrauterine device on the uterine histology and progestational response in pregnant and immature rabbits.

A copper wire intrauterine device (GuIUD) was inserted into mature female rabbits 14 days prior to artificial insemination (AI), and its effect on the histology of the pregnant uterus was studied 48 to 192 hours after AI. In a related study, estrogen-primed immature femal rabbits were fitted with a device consisting of 321 mm of copper wire wound around a 17-mm plastic carrier and treated subcutaneously for 5 days with 0.1 mg of progesterone daily. Uterine tissue samples were obtained on the following day, examined histologically, and tested for carbonic anhydrase activity. In both studies, gold wire devices, similar to the respective copper devices, and sham surgery served as controls. In the pregnant rabbit, the McPhail score (McPhail MK: J Physiol (Lond) 83:145, 1934) measuring arborization of the luminal epithelium was significantly lower in tissue surrounding the CuIUD than in tissue anterior to the CuIUD or in tissue from the contralateral control horn bearing an AuIUD or subjected to sham surgery, 96 to 192 hours after AI. The morphologic development of the epithelial cell structure was retarded only in areas surrounding the CuIUD 96 to 192 hours after AI. There were no observable differences in the stroma or in the leukocytic infiltration of the tissue. In the estrogen-primed immature rabbits, the ability of the tissue to react to exogenous progesterone was depressed in tissue surrounding the copper device, as determined by the lower McPhail score, compared with tissue anterior to the copper device or from contralateral controls. Similary, progestational stimulation of carbonic anhydrase was blocked significantly in tissue surrounding the CuIUD and, to a lesser extent, in tissue anterior to the CuIUD. The data suggest that copper ions eluted from the CuIUD produce a contraceptive effect by locally blocking the action of progesterone on the epithelial tissue.

The effect of a copper intrauterine device during the early stages of pregnancy in the rabbit.

Copper wire intrauterine devices (CuIUD) were surgically inserted into mature female rabbits 15 days prior to artificial insemination. Gold wire intrauterine devices and sham surgery served as controls. Animals were autopsied 48, 72, 96, 120, 144, and 192 hours after artificial insemination. At autopsies performed 48 to 144 hours after insemination, the reproductive tract was flushed and the embryos were examined. At 192 hours, implantation sites were counted and measured. A significant (P less than 0.05) loss of copper from the devices was observed when the devices were weighed after autopsy. The CuIUD had no effect on ovulation, fertilization, or embryo transport up to 120 hours after artificial insemination. The presence of a CuIUD resulted in a slight, but not statistically significant, increase in embryonic degeneration by 120 hours, almost total absence of blastocysts from the uterus by 144 hours, and complete absence of implantation sites 192 hours after insemination. The antifertility effect of a copper intrauterine device appears to occur in the late preimplantation stage of pregnancy in the rabbit.

Uterine copper distribution in monkeys implanted with copper-carrying intrauterine devices.

Chemical and histochemical analyses were carried out on uteri of four monkeys in which plastic IUDs or Cu-IUDs had been implanted for 36 to 43 days. The mean uterine copper content of the plastic-treated animals was 1.1 mug/gm (mean of two), while this value for the Cu-IUD treated monkeys was 1.7 mug/gm. The copper was distributed primarily in the cyclically renewed regions of the endometrium: the luminal fluid, endometrial surface, and superficial lamina propria. The element was localized and was not uniformly distributed in these regions. Copper analyses of plasma, liver, and kidney showed no differences between these two groups. Histopathologic evaluation revealed some areas of edema and increased numbers of neutrophils in plastic-IUD-treated animals. In the Cu-IUD-implanted monkeys, similar changes were observed as was a flattening of the surface epithelium. The endometrium had a loose areolar appearance. The copper elution rate was about 90 mug/day, about twice that observed in women using Cu-IUDs.

Relationship between progesterone suppression and pregnancy in rats.

Four luteolytic agents were administered to groups of pregnant rats to examine the quantitative relationship between serum progesterone levels and the maintenance of pregnancy. Each agent inhibited progesterone in a dose-dependent manner, however only three, azastene, thiosemicarbazone and dihydrotestosterone, adversely affected pregnancy. A statistical analysis of the data suggests that, regardless of the mechanism of action of a particular luteolytic agent, a treatment-induced depression of serum progesterone to concentrations less than 45% of that of the controls on day 11 of pregnancy is incompatible with pregnancy maintenance.

Postcopulatory effects of two antifertility agents on ova transport and implantation.

PIP: The daily doses which prevented implantation in 50 percent of treated animals (ED50) of 2,3 - bis (4-hydroxyphenol) valeronitrile (SC-3402) and 2,3 - bis (4-methoxyphenyl) pent-2-enenitrile (SC-3296) injected in rats s on Days 1 to 3, or Days 4 to 7, or Days 1 to 7 (Day 1 = pregnancy) were 100, 200, and 40 mcg and 50, 100, and 12 mcg respectively, ED50 doses of estrone were 4,8 and 3.5 mcg. Control animals showed ova in the oviduct only on Days 1, 2 and 3, also in the uterus on Day 4, and only in the uterus on Day 5. Very few ova were found in rats treated with 10 mcg estrone daily Day 1-2 and autopsied on Day 3. The same treatment period with 200 mcg SC-3402 caused similar results. 64 mcg SC-3402 resulted in a smaller reduction of ova. Acceleratory potency of 200 mcg SC-3402 is greater than can be due to its estrogenic activity equivalent, 0.5 mcg estrone; that of 64 mcg SC-3296 (4.8 equivalents estrone) can be so ascribed. Rats receiving daily 4-8 mg 17 alpha-acetoxy-6 alpha-methylprogesterone (MAP) from Day 1 to 9 to delay nidation, and 200 mcg SC-3402, autopsied on Day 10 showed no free blastocysts and a few implantation sites in the process of resorption (Free blastocysts were found in rats similarly treated but with ligation of the uterus at the cervix on Day 5 to prevent expulsion of blastocysts). Control rats on Day 10 showed a few implantation sites and free blastocysts. The normal number of implantations were present in SC-3296 treated rats. The average weight of cornu traumatized by threading one cornu in psuedopregnant rats with a silk thread on Day 5 (Day 1=cervical stimulus) in rats treated with 200 mcg SC-3402 on Days 5-8, 404 plus or minus 50 mg was significantly (P less than .05) lower than mean control weight, 794 plus or minus 48 mg. The difference between the mean weight of non-traumatized cornu of rats given 100 mcg, 284 plus or minus 36 and 232 plus or minus 12 mg respectively was significantly (P less than .05) greater than in controls, 159 plus or minus 5.8 mg. The deciduoma-inhibiting activity of SC-3402 is further evidence that it initiates nidation but impedes early implantation stages.^ieng

Biological properties of aspartame. I. Evaluation of central nervous system effects.

Aspartame was administered intragastrically to rodents at doses between 10 and 550 times the expected daily human intake to evaluate the effects on central nervous system function. No biologically meaningful effects were observed in either rats or mice following acute administration by the intragastric route. Aspartame administered as 9% of the diet (about 11 g/kg/day) for thirteen weeks to weanling rats altered the learning behavior of male rats. This effect of impaired learning behavior was nearly identical to that observed for an approximately equimolar amount of L-phenylalanine. The learning behavior of the female rats was not altered by either L-phenylalanine or aspartame at these extremely large doses. It was concluded that prolonged dietary ingestion of aspartame at levels approximately 550 times that expected for normal human daily ingestion was necessary to elicit a behavioral deficit.

The biological properties of aspartame. III. Examination for endocrine-like activities.

A series of studies with aspartame were run in mice, rats and rabbits using standard procedures to characterize possible estrogenic, androgenic, progestational and glucocorticoid activities. Aspartame was administered orally at levels (ca 300 mg/kg/day) substantially in excess of expected maximal human intake when used as a sweetening agent. No significant hormone-mimetic response was observed in the endocrine target organs evaluated. In similar studies, when administered simultaneously with the steroid hormones, it did not reduce the response expected with the steroid. Thus, it was concluded that ingestion of aspartame should not produce any estrogenic, androgenic, progestational or glucocorticoid-like effects. Further, it should not alter the actions of the endogeneous steroid hormones.

11 beta-methyl-19-norsteroids: novel progestational hormones.

PIP: The synthesis and biological properties of 2 11beta-methyl-19-norsteroids (17alpha-ethynyl-17beta-hydroxy-11beta-methylestr-4en-3-one and 3beta, 17beta-diacetoxy-17alpha-ethynyl-11beta-methylestr-4-ene) are described. In experimental animals both compounds exhibited potent progestational activities, antiestrogenic activities, and estrogenic responses in the estrogen-deficient state. It is suggested that the latter compound may be similar to other progestational agents in failing to alter hepatic excretory function and may possess anovulatory properties without some of the side effects associated with steroid contraceptives presently employed.^ieng

The biological properties of aspartame. II. Actions involving the gastrointestinal system.

Aspartame (APM) was investigated in several pharmacological tests to delineate any effects on the gastrointestinal system. The compound did not affect food consumption at one hour following a single intragastric dose of 200 mg/kg in rats. There was no evidence of inhibition or stimulation of the gastric juice secretion rate, the concentration of gastric acid, acid output or proteolytic activity following an intragastric dose of 250 mg/kg in five-hour pylorus-ligated rats. Likewise, APM at the same dosage did not significantly affect gastric ulceration induced by nineteen hours of pylorus-ligation. In several in vitro tests it was demonstrated that APM did not affect the proteolytic activity of pepsin or the lipolytic activity of pancreatic lipase at concentrations of 143 microgram and 1.25 mg/ml, respectively. Its anticholinergic activity was found to be insignificant, less than 0.001 times the potency of atropine sulfate, when measured against acetylcholine-induced contraction of isolated rabbit ileum. These data indicate that APM may be devoid of undesirable side effects on the gastrointestinal tract when used as a food sweetening agent.

The biological properties of aspartame. V. Effects on a variety of physiological parameters related to inflammation and metabolism.

Aspartame (APM), L-aspartyl-L-phenylalanine methyl ester, is a low calorie sweetening agent 180 times sweeter than sucrose. As part of a series of studies designed to determine the potential effects of ingestion of excesses of APM on a wide spectrum of physiological processes, experiments were conducted in which high multiples (mg/kg basis) of the projected maximum daily human intake (20 mg/kg) were administered intragastrically to laboratory rats. Doses up to 16 times the maximum intake had no effect on inflammation parameters including carrageenin-induced paw edema, connective tissue formation and adjuvant arthritis. APM, likewise, showed no antihistamine activity in vitro. Even higher multiples (up to 103 times) of the maximum intake had no effect on various parameters of carbohydrate and lipid metabolism. These results indicate that APM ingested in great excess would not be expected to significantly impair inflammatory processes nor influence carbohydrate and lipid metabolism.

The biological properties of aspartame. IV. Effects on reproduction and lactation.

Intragastric administration of approximately 300 mg/kg/day of aspartame (APM) to female rats for seven days and to female hamsters for five days after mating did not affect postcoital fertility as measured by the number of implantation sites and normal appearing fetuses. In additional studies, the effect of APM fed at 1 to 14% in the diet to lactating rats and their litters of suckling young was studied using a pair-feeding experimental design. Levels of APM up to 4% in the diet (about 7 g/kg/day) did not affect food consumption, body weights, serum prolactin, serum gonadotropins, the mammary gland histology of the dams or the growth and survival rates of their pups. However, higher levels of 7.5 and 14% APM (about 9 g/kg/day) caused reduced food consumption due to diet palatability and resulted in body weight loss in dams and retarded growth rates in the young.