Jerry R. Reel

Reproductive effects of four phthalic acid esters in the mouse

These studies compared the reproductive toxicity of four phthalates by a continuous breeding protocol. Mice were given diets with diethyl phthalate (DEP) (0.0, 0.25, 1.25, or 2.5%), di-n-butyl phthalate (DBP) (0.0, 0.03, 0.3, or 1.0%), di-n-hexyl phthalate (DHP) (0.0, 0.3, 0.6, or 1.2%), or di(2-ethylhexyl) phthalate (DEHP) (0.0, 0.01, 0.1, or 0.3%). Both male and female CD-1 mice were dosed for 7 days prior to and during a 98-day cohabitation period. Reproductive function was evaluated during the cohabitation period by measuring the numbers of litters per pair and of live pups per litter, pup weight, and offspring survival. There was no apparent effect on reproductive function in the animals exposed to DEP, despite significant effects on body weight gain and liver weight. DBP exposure resulted in a reduction in the numbers of litters per pair and of live pups per litter and in the proportion of pups born alive at the 1.0% amount, but not at lower dose levels. A crossover mating trial demonstrated that female mice, but not males, were affected by DBP, as shown by significant decreases in the percentage of fertile pairs, the number of live pups per litter, the proportion of pups born alive, and live pup weight. DHP in the diet resulted in dose-related adverse effects on the numbers of litters per pair and of live pups per litter and proportion of pups born alive at 0.3, 0.6, and 1.2% DHP in the diet. A crossover mating study demonstrated that both sexes were affected. DEHP (at 0.1 and 0.3%) caused dose-dependent decreases in fertility and in the number and the proportion of pups born alive. A crossover mating trial showed that both sexes were affected by exposure to DEHP. These data demonstrate the ability of the continuous breeding protocol to discriminate the qualitative and quantitative reproductive effects of the more and less active congeners as well as the large differences in reproductive toxicity attributable to subtle changes in the alkyl substitution of phthalate esters.

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

In vitro antiprogestational/antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone.

In determining the biological profiles of various antiprogestins, it is important to assess the hormonal and antihormonal activity, selectivity, and potency of their proximal metabolites. The early metabolism of mifepristone is characterized by rapid demethylation and hydroxylation. Similar initial metabolic pathways have been proposed for CDB-2914 (CDB: Contraceptive Development Branch of NICHD) and CDB-4124, and their putative metabolites have been synthesized. We have examined the functional activities and potencies, in various cell-based assays, and relative binding affinities (RBAs) for progesterone receptors (PR) and glucocorticoid receptors (GR) of the putative mono- and didemethylated metabolites of CDB-2914, CDB-4124, and mifepristone and of the 17alpha-hydroxy and aromatic A-ring derivatives of CDB-2914 and CDB-4124. The binding affinities of the monodemethylated metabolites for rabbit uterine PR and human PR-A and PR-B were similar to those of the parent compounds. Monodemethylated mifepristone bound to rabbit thymic GR with higher affinity than monodemethylated CDB-2914 or CDB-4124. T47D-CO cells were used to assess inhibition of R5020-stimulated endogenous alkaline phosphatase activity and transactivation of the PRE(2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid in transient transfections. The antiprogestational potency was as follows: mifepristone/CDB-2914/CDB-4124/monodemethylated metabolites (IC(50)s approximately 10(-9)M) > aromatic A-ring derivatives (IC(50)s approximately 10(-8)M) > didemethylated/17alpha-hydroxy derivatives (IC(50)s approximately 10(-7)M). Antiglucocorticoid activity was determined by inhibition of dexamethasone-stimulated transcriptional activity in HepG2 cells. The mono- and didemethylated metabolites of CDB-2914 and CDB-4124 had less antiglucocorticoid activity (IC(50)s approximately 10(-6)M) than monodemethylated mifepristone (IC(50) approximately 10(-8)M) or the other test compounds. At 10(-6)M in transcription assays, none of these compounds showed progestin agonist activity, whereas mifepristone and its monodemethylated metabolite manifested slight glucocorticoid agonist activity. The reduced antiglucocorticoid activity of monodemethylated CDB-2914 and CDB-4124 was confirmed in vivo by the thymus involution assay in adrenalectomized male rats. The aromatic A-ring derivatives-stimulated transcription of an estrogen-responsive reporter plasmid in MCF-7 and T47D-CO human breast cancer cells but were much less potent than estradiol. Taken together, these data suggest that the proximal metabolites of mifepristone, CDB-2914, and CDB-4124 contribute significantly to the antiprogestational activity of the parent compounds in vivo. Furthermore, the reduced antiglucocorticoid activity of CDB-2914 and CDB-4124 compared to mifepristone in vivo may be due in part to decreased activity of their putative proximal metabolites.

Disruption of Spermatogenesis and Sertoli Cell Structure and Function by the Indenopyridine CDB-4022 in Rats

Abstract The present studies were undertaken to determine the testicular cell type(s) affected by the antispermatogenic indenopyridine CDB-4022. At the oral threshold dose (2.5 mg/kg), CDB-4022 induced infertility in all males. CDB-4022 did not alter (P > 0.05) Leydig cell function as assessed by circulating testosterone, seminal vesicle, and ventral prostate weights or body weight gain compared to controls. Conversely, CDB-4022 reduced (P < 0.05) testicular weight, spermatid head counts, and percentage of seminiferous tubules undergoing spermatogenesis. In a second study, adult male rats received a maximally effective oral dose of CDB-4022 (12.5 mg/kg), dipentylphthalate (DPP; 2200 mg/kg; a Sertoli cell toxicant), or vehicle and were necropsied 3, 6, or 12 h after dosing to determine acute effects. Serum inhibin B levels were suppressed (P < 0.05) by 6 h after CDB-4022 or DPP treatment, but epididymal androgen-binding protein (ABP) levels were not altered (P > 0.05), compared to controls. CDB-4022 and DPP increased (P < 0.05) the percentage of tubules with apoptotic germ cells, particularly differentiating spermatogonia and spermatocytes, by 12 h after dosing. Microscopic examination of the testis indicated a greater degree of vacuolation in Sertoli cells and initial signs of apical germ cell sloughing/shedding by 3 or 12 h after CDB-4022 or DPP treatment, respectively. In a third study, prepubertal male rats were treated with vehicle, 12.5 mg/kg of CDB-4022, or 2200 mg/kg of DPP, and the efferent ducts of the right testis were ligated 23 h before necropsy. Seminiferous tubule fluid secretion (difference in weight of testes), serum inhibin B levels, and ABP levels in the unligated epididymis were reduced (P < 0.05) at 24 and 48 h after dosing in CDB-4022- and DPP-treated rats compared to controls. Collectively, these data suggest that CDB-4022 disrupts spermatogenesis by inducing apoptosis in early stage germ cells via a direct action on the Sertoli cell.

Antiovulatory and postcoital antifertility activity of the antiprogestin CDB-2914 when administered as single, multiple, or continuous doses to rats

The present studies in rats were undertaken to investigate the potential of a new antiprogestin, CDB-2914, for use as an emergency postcoital contraceptive for women. When given orally at noon on the day of proestrus, both CDB-2914 and mifepristone displayed dose-dependent antiovulatory activity; however, CDB-2914 was about eight times more potent than mifepristone. Both antiprogestins were considerably less potent in blocking ovulation when injected subcutaneously. To evaluate antifertility activity during continuous low dose administration, rats were dosed orally with 0.5 mg of either CDB-2914 or mifepristone daily, commencing on the day of estrus and continuing for 24 days. Females were cohabited with proven fertile males on day 8 of treatment and were removed 1-3 days later after confirmed mating. The pregnancy rate was significantly reduced (p < 0.05) only in the CDB-2914-treated females; however, the mean number of normal implantation sites per pregnant rat was significantly reduced (p < 0.05) by mifepristone as compared with the vehicle control group. CDB-2914 was also found to prevent pregnancy when administered orally after mating from days 0-3 during tubal egg transport, or from days 4-6 during the pre- and peri-implantation periods. To determine the day of maximal sensitivity to CDB-2914, a single 2-mg dose per rat was given orally on days 0, 1, 2, 3, 4, or 5 postmating. This dose of CDB-2914 was without effect on pregnancy at days 0, 1, 2, or 3 postmating. In contrast, 2 mg CDB-2914 per rat was highly effective in blocking pregnancy when given on either day 4 or 5 postmating. Collectively, these data demonstrate that CDB-2914 is an orally active postcoital antifertility agent that is more potent than mifepristone in the rat. Hence, CDB-2914 may prove to be an effective emergency postcoital contraceptive in women.

The Ability of a Gonadotropin-Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone

Abstract Intratesticular testosterone (ITT) is known to play a critical role in the maintenance of spermatogenesis. We have used acyline, a GnRH antagonist, to suppress testosterone (T) production, and acyline and T implants to study the prevention of irreversible infertility induced by CDB-4022. Vehicle or acyline was administered to proven fertile male rats (n = 5/group) at a dose (210 μg/day) that completely suppressed (P < 0.05) T production, as measured by serum T, and testicular function, either before, concurrent with, or after vehicle or a single oral dose of 2.5 mg CDB-4022/kg (Week 0). Vehicle-treated males remained fertile, whereas acyline-treated males exhibited transitory infertility. CDB-4022 alone caused irreversible infertility in all males. Importantly, CDB-4022-treated males recovered fertility when acyline was started before CDB-4022 (Weeks −4 to 0; Weeks −4–9), but not when acyline was administered concurrently with or after CDB-4022 (Weeks 0–9; Weeks 10–19). At the end of this study (Week 34), testes weights, spermatid head counts (SHC), and tubule differentiation indices (TDI) were suppressed (P < 0.05) in infertile CDB-4022-treated males, but in rats that recovered fertility, these parameters were similar (P > 0.05) to those in vehicle-treated males. In addition, serum inhibin B and epididymal androgen-binding protein levels were nondetectable in infertile CDB-4022-treated rats. To test whether suppression of ITT was critical for prevention of CDB-4022-induced infertility, proven fertile rats (n = 7–8/group) received vehicle, acyline alone, or acyline and a T implant for 4 wk before CDB-4022 (Week 0). The T implant increased ITT in acyline-treated rats. Although ITT was lower (P < 0.05) in the T-implanted males than in untreated rats, it was sufficient to sustain spermiogenesis. Serum rFSH levels were also elevated in rats treated with acyline + T as compared with acyline alone during the treatment interval, but rFSH was still lower than in vehicle-treated rats. Rats in all treatment groups were rendered infertile initially, but the acyline + CDB-4022-treated rats recovered fertility by Week 10. In contrast, rats treated with CDB-4022 alone or acyline + T + CDB-4022 remained infertile until at least Week 16. Testes weights, SHC, and TDI were within normal ranges for acyline + CDB-4022-treated rats, but were decreased (P < 0.05) in CDB-4022- or acyline + T + CDB-4022-treated rats. Serum inhibin B levels were nondetectable by Week 1 in males rendered irreversibly infertile by CDB-4022; levels increased transiently and returned to baseline in rats protected by acyline pretreatment. These data indicate that pretreatment with acyline was able to prevent irreversible infertility in CDB-4022-treated rats, whereas posttreatment with acyline did not promote spermatogonial differentiation, as has been observed by others in rats that received GnRH analogs and various other testicular toxicants. Suppression of ITT and possibly rFSH by acyline appeared to be crucial in preventing irreversible infertility induced by CDB-4022. In this regard, our results are similar to those of investigators who have studied other testicular toxicants. Continued development of CDB-4022 as a potential male contraceptive will depend largely on its safety profile and whether its antispermatogenic activity is reversible in primates.

Lupron Depot Prevention of Antispermatogenic/Antifertility Activity of the Indenopyridine, CDB-4022, in the Rat

Abstract The goals of this study were to determine the CDB-4022 dose-response relationship for induction of acute decreases in testicular weight and germ cell depopulation in rats; establish the threshold dose of CDB-4022 required to induce infertility; and investigate whether CDB-4022-induced testicular damage could be prevented by a GnRH agonist (Lupron Depot). Reduction of testis weight and germ cell depopulation were observed 7 days after a single oral dose of 1 mg CDB-4022/kg, whereas 0.5 mg/kg had no observable effect. These effects were maximal at 12.5 or 25 mg CDB-4022/kg. After a single oral dose of either 2.5 or 5 mg/kg, CDB-4022 induced infertility in five of five treated rats by Week 5, whereas only one of five males was rendered infertile at a dose of 1 mg/kg. Proven fertile male rats (6/group) were treated with vehicle, CDB-4022 alone (2.5 mg/kg on Day 0), CDB-4022 plus Lupron Depot (on Weeks −1, 2, 5, and 8), or Lupron Depot alone. Control males demonstrated normal fertility throughout a 32-wk cohabitation period. Five of six rats were rendered transiently infertile with Lupron Depot alone, but all recovered fertility. CDB-4022 treatment resulted in infertility in all six rats, and only one of six regained fertility. Combined treatment also caused infertility in all six rats, but four of six recovered fertility (P = 0.08 compared to CDB-4022 alone). Testicular weight was decreased in the three treatment groups compared to vehicle controls; testicular weights were ranked from highest to lowest as follows: vehicle > Lupron Depot > Lupron Depot + CDB-4022 > CDB-4022. The tubule differentiation index of Lupron Depot-treated rats (96 ± 4%) was not different from vehicle-treated rats (100%). CDB-4022 treatment decreased the number of differentiating tubules (15 ± 8%). Lupron Depot plus CDB-4022 treatment resulted in a greater number of differentiating tubules (53 ± 12%) than CDB-4022 alone, but this was still lower than vehicle- or Lupron Depot-treated rats. These data indicate that 2.5 mg/kg of CDB-4022 was the oral threshold dose that caused testicular damage rendering the majority of adult male rats permanently infertile within the study interval; 12.5 mg/kg of CDB-4022 induced maximal testicular damage. Suppression of gonadotropins and/or testosterone production by treatment with Lupron Depot before and after CDB-4022 prevented the CDB-4022-induced irreversible testicular damage.

Steroid hormonal regulation of growth, prostate specific antigen secretion, and transcription mediated by the mutated androgen receptor in CWR22Rv1 human prostate carcinoma cells

CWR22Rv1 (22Rv1) is an androgen-responsive human prostate carcinoma cell line derived from a primary prostate tumor that expresses mutant (H874Y) androgen receptors (AR) and secretes low levels of prostate specific antigen (PSA). In this study, we examined the effects of various androgens and other steroid hormones on proliferation of 22Rv1 cells, PSA secretion, and transactivation. Incubation of 22Rv1 cells with various concentrations of testosterone resulted in a dose-dependent 50-80% increase in growth over 72 h. PSA release and transactivation of PRE2-tk-LUC in 22Rv1 cells were stimulated by low concentrations of natural and synthetic androgens (EC(50)s = 10(-10) to 10(-9)M) and a broad range of other classes of steroid hormones, albeit with lower potency. Uniform positive immunocytochemical staining was observed in 22Rv1 cell nuclei with mouse monoclonal antibodies to human AR. Competitive binding assays indicated that the mutant AR in 22Rv1 cytosol is more promiscuous than a wild-type AR (ARLBD: rat AR ligand binding domain). Testosterone (10(-8)M)-induced PSA release and transactivation were blocked by both antiandrogens and antiprogestins with IC(50)s of 10(-7) to 10(-6)M. At high concentration (10(-6)M), these antagonists showed partial agonist activity in terms of PSA secretion but not transactivation. In conclusion, the mutant AR in 22Rv1 cells binds and responds to low levels of androgens and a wide spectrum of other natural and synthetic steroid hormones, mechanisms proposed to contribute to tumor progression following androgen ablation.

Evaluation of a New Reproductive Toxicology Protocol Using Diethylstilbestrol (DES) as a Positive Control Compound

A new National Toxicology Program (NTP) reproductive toxicology assay designated Fertility Assessment by Continuous Breeding was evaluated using trans-diethylstilbestrol (DES) as a positive control compound. The testing scheme employs young adult CD-1 male and female mice and is comprised of 5 possible tasks: task 1, 14-day dose-finding (repeated or continuous dosing); task 2, continuous breeding; task 3, determination of affected sex; task 4, offspring assessment; task 5, hormone patterns. Only tasks 2 and 3 were performed in the present study. Task 2 employed dietary levels of 0, 1, 10, and 50 ppb DES (99% pure). Task 3 used the control and high dose parental mice from task 2 in a crossover mating trial (control x treated) to determine whether one or both sexes in the F0 generation were adversely affected. Continuous exposure of F0 mice to 50 ppb DES in the diet during task 2 significantly decreased (P < 0.01) the proportion of pairs that produced at least 1 litter as compared to pairs continuously exposed to dietary levels of 0, 1, and 10 ppb DES. In addition, continuous exposure to 50 ppb DES in the diet had a cumulative adverse effect on the production and viability of litters. Thus, the pairs fed 50 ppb DES produced significantly fewer litters (P < 0.01), had fewer live pups per litter (P < 0.01), and tended toward a lower proportion of pups born alive per litter than did pairs in the 0, 1, and 10 ppb DES groups. In the crossover mating trial (task 3) the proportion of F0 females that mated (number with copulatory plugs/number cohabited) and that were fecund (number delivering litters/number with copulatory plugs) did not differ significantly among the 3 different combinations of paired mice. It was noted, however, that the proportion of cohabited F0 females delivering litters (number delivering litters/number cohabited) was significantly reduced (P < 0.05) in the control male x 50 ppb DES female group vs. the control male x control female group. The proportion of cohabited F0 females delivering litters in the 50 ppb DES male x control female group did not differ significantly from either of the above groups. Further, the number of live pups per litter and the proportion of pups born alive were significantly lower (P < 0.01) in the F0 control male x 50 ppb DES female group than in the other 2 groups. The proportion of male pups born alive (males/total) and the live pup weights, however, did not differ significantly among the 3 combinations of paired mice. Collectively, these results indicated that DES interfered with ovulation, conception, pre- and postimplantational processes in female mice. Thus, this new assay readily discriminated DES as a reproductive toxicant in CD-1 mice.

Reproductive Toxicity Evaluation of Acetaminophen in Swiss CD-1 Mice Using a Continuous Breeding Protocol

Acetaminophen (APAP) was evaluated for reproductive toxicity in Swiss CD-1 mice using a continuous breeding protocol. APAP was administered in the diet at 0, 0.25, 0.5, and 1.0% (w/w), which represented average daily intakes of 0, 357, 715, and 1430 mg APAP/kg/day, respectively. Exposure of parental (P) breeding pairs to 1% APAP in the diet for 14 weeks during cohabitation significantly decreased the number of litters per pair, and reduced, although not significantly, the number of live pups per litter. Importantly, 6 of 19 high-dose P pairs failed to produce a fifth litter, and this fully accounted for the diminished number of litters in this group. In addition, the fifth litter that was produced by the 13 high-dose P pairs averaged only about 9 live pups per litter, which correspondingly reduced the overall group average for this parameter. In comparison, the control and two lower-dose P pairs produced 11 or 12 live pups per litter on average. Although the birth weights for F1 pups in the final litter were unaffected by prenatal APAP exposure, postnatal growth was adversely affected as evidenced by retarded weight gain as measured at 28 and 74 +/- 10 days of age for all three dietary levels. At 1% APAP this weight gain effect was more pronounced at Day 28 than at Day 74 +/- 10, suggesting that nursing pups may have been exposed to higher concentrations or may be more sensitive to APAP and/or an active metabolite than were the young adults.(ABSTRACT TRUNCATED AT 250 WORDS)