Lack of biologically active estrogens in commercial cow milk.

Recently, many studies have investigated potential estrogenic compounds in the human diet. Several of these investigations have studied cow milk, a mainstay of the diets of both young and old. In vitro studies have determined that estrogens can be found in milk, and that the concentration of estrogen may be correlated to the fat content in the milk. Regardless, the majority of these studies have concluded that the levels of estrogens in milk are too low to have a physiological effect. However, a recent study found that commercial 1% cow milk was uterotrophic in rats, suggesting that it contained biologically significant levels of estrogen. Using the rat model, we tested milk samples from commercial sources and with varying fat content for estrogenic activity. Ovariectomized female rats were given milk ad libitum for a period of 2 wk. After 12 d of treatment, rats were tested sequentially in an open field and an elevated plus maze to determine any effect of milk on anxiety levels. Upon completion of the behavior testing, uterine weights were examined. Regardless of milk type, no difference was observed in daily volume of milk consumed. Contrary to previous publications, no differences existed in either the behavior or the uterine weights between animals that consumed any milk type and the negative controls. These results demonstrated that none of the commercial milk types that we tested contained biologically significant estrogenic activity.

Developmental and tissue-specific expression of human CD4 in transgenic rabbits.

A major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV-1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hcD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952-2958). The hCD4 transgene was detected in five males and two females derived from the microinjection in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T-cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4- rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV-1 p24 core protein following HIV-1 infection in vitro than HIV-1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high-level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV-1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV-1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV-1 infection. In vivo HIV-1 infection studies with these rabbits are under way.

Non-steroidal glucocorticoid-like substances: receptor binding and in vivo activity.

Compounds of general structure I, prepared by a Diels-Alder reaction with diene 3, are relatives of the known potent glucocorticoid II but possess a markedly modified C- and D-ring environment. Despite these structural changes, 4, 5, 9, 10, 12a, 13, and 14 bound to the glucocorticoid receptor with an affinity which approximated that of the reference standard, 6-alpha-methylprednisolone. Four of these compounds not only exhibited antiinflammatory activity in the alpha-tocopherol pouch test but also exhibited marked adrenal suppression and other typical glucocorticoid properties at doses in the same range as the effective antiinflammatory doses.

Tissue-specific expression of human CD4 in transgenic mice.

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines.

Antiandrogenic steroidal sulfonylpyrazoles.

The steroidal sulfonylpyrazole 1 bound to the rat ventral prostate androgen receptor in vitro; it inhibited testosterone propionate induced increases in ventral prostate weight in vivo in the castrated, immature male rat with an ED50 of 15 mg/kg po. Compound 1 lacked androgenic activity in vivo in contrast to the parent steroidal pyrazole 5, which was both androgenic and antiandrogenic. The 2'- and 5'-methylsulfonyl isomers 6 and 6a did not bind to the androgen receptor. Introduction of an alkylsulfonyl at the N-1'-position has served, therefore, to isolate the intrinsic antiandrogenic properties of the steroidal heterocycle free of apparent hormone agonist properties. Structure-activity relationship studies revealed that a methylsulfonyl group at N-1' together with a C-17 alpha-substituent were the optimal combination for in vitro androgen receptor binding, in vivo antiandrogenic potency, and a lack of androgenic activity.

Danazol suppression of luteinizing hormone in the rat: evidence for mediation by both androgen and estrogen receptors.

Previous studies had indicated that the effects of danazol on rat peripheral tissues were mediated through both androgen and estrogen receptors. The purpose of this study was to examine the role of androgen and estrogen receptors in danazol suppression of luteinizing hormone (LH) in the rat. The estrogen receptor antagonist, LY 156758, partially antagonized the suppressed levels of LH observed 3-5 hr after administration of danazol to ovariectomized rats. In contrast, the androgen receptor antagonist, flutamide, had no effect on suppressed LH levels 3-5 hr after danazol, but did partially reverse the inhibition of LH 24 hr after danazol administration to ovariectomized rats. Danazol also increased pituitary progesterone receptor levels, an estrogen-sensitive end point. These data indicate that the suppressed levels of LH following danazol treatment resulted from the functional activation of both androgen and estrogen receptors.

Endocrine profile of Win 49596 in the rat: a novel androgen receptor antagonist.

Win 49596 is a new orally active, steroidal androgen receptor antagonist. Win 49596 inhibited ventral prostate, seminal vesicle and levator ani weight gain in either 5 alpha-dihydrotestosterone (DHT) or testosterone propionate-treated castrated, immature male rats. In intact, adult male rats, Win 49596 significantly inhibited weight gain by the ventral prostate, dorsal lateral prostate and seminal vesicles, but not the testes at doses as low as 50 mg/kg/day x 14 p.o. However, daily oral administration of equivalent antiandrogenic doses of either Win 49596, ICI 176,334, or flutamide for 14 days to mature, intact male rats resulted in elevations of circulating testosterone of approximately 3-, 2-, and 10-fold, respectively. At doses as high as 400 mg/kg p.o., Win 49596 did not have androgenic, progestational, estrogenic or antiestrogenic activity in rat or rabbit models. However, in the Clauberg assay, Win 49596 did have weak antiprogestational activity at doses of 25-400 mg/kg/day p.o. These data indicate that Win 49596 is a peripherally selective antiandrogen that has minimal effects on circulating testosterone levels and is devoid of hormone agonist activity. Thus, Win 49596 may be useful for the treatment of androgen dependent conditions such as benign prostatic hyperplasia and prostatic cancer.

Antiandrogens do not reverse androgen-induced inhibition of sex hormone-binding globulin levels in adult female rhesus monkeys.

Studies were done to determine if changes in plasma sex hormone-binding globulin (SHBG) levels could serve as a specific marker of androgenic and antiandrogenic activities in rhesus monkeys. Treatment of adult female monkeys for 11 days with 17 alpha-methyltestosterone (MeT) produced dose and time-dependent reductions in SHBG levels. However, the non-steroidal antiandrogen flutamide (80 mg/monkey.day) did not inhibit the reduction in SHBG levels when coadministered with MeT, nor did it have an effect on SHBG levels when given alone. In contrast, the steroidal antiandrogens Win 49596 (100 mg/monkey.day) and cyproterone acetate (80 mg/monkey.day) significantly (P less than 0.01) reduced SHBG plasma concentration to about 50% of pretreatment control values whether given alone or in combination with MeT. Furthermore, Win 49596 reduced SHBG levels at doses as low as 4 mg/monkey, whereas cortico-steroid-binding globulin levels were not affected. In ovariectomized monkeys, MeT treatment (4 mg/monkey.day for 15 days) reduced plasma SHBG levels to 42% of pretreatment values and delayed the onset of withdrawal menstrual bleeding compared to that in controls. When administered concurrently with MeT, flutamide (100 mg/monkey.day) antagonized the effect on withdrawal bleeding, but was without effect on SHBG levels. Therefore, plasma SHBG levels cannot be used as a specific indicator of androgenic or antiandrogenic activity and may not be regulated through the classical androgen receptor.

Studies on the mechanism of action of danazol and gestrinone (R2323) in the rat: evidence for a masked estrogen component.

The effects of androgen and estrogen receptor antagonists on the action of danazol and gestrinone (R2323) were investigated. The tropic effect of danazol and gestrinone on sex accessory tissues of castrated, immature male rats was inhibited by the antiandrogen flutamide, whereas the uterotropic action of these steroids in immature female and adult ovariectomized rats was not inhibited by flutamide. In contrast, the uterotropic effect of danazol was reduced by the antiestrogen, LY156758. The estrogen-sensitive endpoints, vaginal keratinization and uterine progesterone receptor concentration, were enhanced by treatment with a combination of flutamide and either danazol or gestrinone. These data indicate that danazol and gestrinone have estrogenic activity that is masked by the androgenic component of these drugs.

Effect of epostane, ZK 98299, and ZK 98734 on the interruption of pregnancy in the rat.

The objective of these studies was to determine whether treatment of 10-day pregnant rats with a combination of epostane (a progesterone biosynthesis inhibitor) and either ZK 98299 or ZK 98734 (progesterone receptor antagonists) would result in additive or synergistic effects on the interruption of pregnancy. When these compounds were tested individually, the order of potency in interrupting pregnancy was ZK 98734 greater than ZK 98299 greater than epostane (50% effective doses 1.3, 4.0, and 35 mg/kg, respectively). Epostane and ZK 98299 were then tested in combination. When epostane was given either 4 h prior to or concurrently with ZK 98299, the combined drug treatment resulted in a significant additive increase in interceptive activity compared to when ZK 98299 was administered alone. In vitro binding studies showed that ZK 98299 and ZK 98734 bound to the rat uterine progesterone receptor in vitro with approximately equal affinity. ZK 98734 bound to the rat thymus glucocorticoid receptor and to the rat ventral prostate androgen receptor with a greater affinity than ZK 98299. The affinity of ZK 98299 for the rat uterine estrogen receptor was weak while the binding of ZK 98734 was not detectable. Thus, the in vitro receptor binding profiles observed were consistent with the known progesterone and glucocorticoid antagonist activities of ZK 98299 and ZK 98734. Overall these findings show that the interceptive activity of epostane and ZK 98299, agents that exert their interceptive activity via different molecular mechanisms, is additive in the 10-day pregnant rat.

The effect of danazol in the MRL/lpr mouse model of autoimmune disease.

The purpose of the study was to determine if danazol was efficacious in the treatment of lupus MRL/MpJ (lpr) mice, as measured by longevity, proteinuria and serum amyloid protein (SAP) levels. Danazol, administered at an oral dose of 100 mg/kg, significantly prolonged survival of female MRL/MpJ (lpr) mice but had no effect on the mortality of their male counterparts. Medication with danazol began 40 days after birth of the mice and resulted in a significant decrease in proteinuria in female but not male lupus mice. The concentration of SAP, an acute phase reactant, was significantly decreased in danazol-treated female lupus mice at 80, 100, 120, 140 and 160 days of age when compared to vehicle-treated control mice. SAP levels in male lupus mice treated with danazol were significantly lower than normal control levels only at the 120 and 160 day time points. Measurements of mortality, proteinuria and SAP concentration indicate that danazol at 100 mg/kg is orally active in the treatment of MRL/MpJ (lpr) female, but not male mice.

The effect of glucocorticoids on plasma fibronectin levels in normal and arthritic rats.

In vivo studies with normal and adjuvant-induced arthritic rats were undertaken in order to measure the effects of glucocorticoids on paw inflammation and plasma fibronectin (Fn) levels. Dexamethasone, methylprednisolone, and corticosterone all enhanced plasma Fn levels in normal animals. All drugs also significantly decreased inflammation in arthritic rats as measured by paw swelling. Of the three glucocorticoids, only corticosterone did not significantly enhance Fn levels in arthritic rats, possibly due to its lesser potency and narrow therapeutic window.

Inhibition of luteal phase progesterone levels in the rhesus monkey by epostane.

This study examined the effect of epostane, a new antifertility drug, on normal and hCG-stimulated progesterone production during the luteal phase of the menstrual cycle in rhesus monkeys. When administered once each day for five days, epostane inhibited normal luteal phase progesterone levels in a dose-related fashion. Epostane also reduced the elevated luteal phase progesterone levels of animals treated with hCG indicating that the drug acts directly on the corpus luteum. These data suggest that epostane interferes with corpus luteum function in a primate and that the drug would be effective as an interceptive agent during early pregnancy.

Glucocorticoid/antiinflammatory activities of nonsteroidal phenylpyrazoles in the rat.

Six nonsteroidal phenylpyrazoles are described that have significant glucocorticoid and antiinflammatory activities. These agents competed with dexamethasone for the glucocorticoid receptor from the rat thymus, suppressed adrenal weight when administered orally to intact female rats, produced liver glycogen deposition and thymolysis when administered orally to adrenalectomized male rats, and reduced cotton granuloma formation when administered in the cotton pellet. In addition, in the latter model, no systemic activity (thymolysis or reduced body weight gain) was seen with doses up to 500 to 5000 times the dose which reduced granuloma formation. At least one compound was more potent than methylprednisolone in three of the four rat assay systems used. The compounds described are structurally different from conventional steroidal glucocorticoids but possessed potent glucocorticoid activities. However, they exhibited antiinflammatory activity without evidence of systemic activity when administered locally.

Inhibition of ovulation in rats with epostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase.

Epostane (Win 32729), an inhibitor of the 3 beta-hydroxysteroid dehydrogenase enzyme system, inhibited both spontaneous and pregnant mare's serum/human chorionic gonadotropin-induced ovulation in rats. When administered on the morning of proestrus, the drug blocked pregnancy in females that were inseminated that evening. The blockage of pregnancy occurred at a dose of 200 mg/kg but not at 50 mg/kg. Similarly, when administered on the morning of proestrus at a dose of 200 mg/kg, epostane inhibited the appearance of ova in the oviducts the next day. In gonadotropin-primed immature rats, epostane inhibited ovulation in a dose-related fashion with an ED50 between 25 and 50 mg/kg. The drug also decreased plasma progesterone levels in these animals. The inhibitory effect of epostane on gonadotropin-stimulated ovulation was reversed by injections of progesterone at a total daily dose of 6.25 mg/rat or greater. These results support the contention that steroidogenesis, specifically progesterone synthesis, is a prerequisite to ovulation.

Nivazol: a glucocorticoid in rats with only hypothalamic-pituitary-adrenal-inhibiting activity in primates.

The synthetic steroid nivazol lacks three of the substituents considered to be important for glucocorticoid activity, i.e. the 3-keto, the 11-hydroxy, and the 20-keto groups. Nevertheless, in the rat, nivazol has the activity profile of a glucocorticoid. After treatment of intact female rats with nivazol , the mean weights of the adrenals and thymus were lower than those in the vehicle-treated control group. The results were qualitatively and quantitatively similar to those obtained with methylprednisolone. Thymolysis as well as liver glycogen deposition were seen in adrenalectomized rats treated with nivazol , and eosinopenia and inhibition of carrageenan edema were noted in intact rats. In the rhesus monkey, treatment with nivazol resulted in a marked reduction in circulating cortisol levels and elimination of the diurnal pattern, although a dose 10 times that needed to reduce circulating cortisol levels did not produce eosinopenia or increase fasting blood glucose levels. Both eosinopenia and higher fasting blood glucose levels were seen after treatment with methylprednisolone. Nivazol did not prevent the ACTH-induced increase in circulating cortisol levels nor did it alter circulating aldosterone levels. Therefore, suppression of ACTH is the predominant if not the sole action of nivazol in the primate. Preliminary results in clinical trials suggest a similar activity profile in humans. Therefore, nivazol elicits numerous glucocorticoid activities in the rodent, but only the inhibition of the hypothalamic-pituitary-adrenal axis was observed in the primate. It is of special interest that the inhibition of the hypothalamic-pituitary-adrenal axis occurs without altering circulating aldosterone levels and without evidence of debilitating catabolic activity.

Estradiol-17 beta interference with meiotic maturation in Rana pipiens ovarian follicles: evidence for inhibition of 3 beta-hydroxysteroid dehydrogenase.

Increasing doses of estradiol-17 beta added to in vitro incubations inhibited pregnenolone-induced germinal vesicle breakdown in Rana pipiens ovarian follicles. The inhibition was reversed with increasing concentrations or pregnenolone added to the medium. Because no evidence of estradiol-17 beta inhibition or interaction with progesterone-induced GVBD was observed, the effect of estradiol-17 beta on the conversion of 3H-pregnenolone to 3H-progesterone was investigated. Estradiol-17 beta in doses as low as 10(-7) M significantly inhibited the conversion of 3H-pregnenolone to 3H-progesterone in follicles incubated in vitro. It is suggested that estradiol-17 beta is a feedback inhibitor of 3 beta-hydroxysteroid dehydrogenase-isomerase, the enzyme complex that converts pregnenolone to progesterone, a necessary step in the initiation of GVBD.