Male Reproductive Tract Malformations in Rats Following Gestational and Lactational Exposure to Di(n-butyl) Phthalate: An Antiandrogenic Mechanism?

Di(n-butyl) phthalate (DBP), a widely used plasticizer suspectedof having estrogenic properties, was investigated for its effectson the prenatal and early neonatal development of the reproductivetract. Pregnant CD rats (n = 10) were given DBP at 0, 250, 500,or 750 mg/kg/day (p.o.) throughout pregnancy and lactation untiltheir offspring were at postnatal day 20. Maternal body weightsthroughout the dosing period were comparable in all groups.At 750 mg/kg/day, the number of live pups per litter at birthwas decreased and maternal effects on pregnancy and postimplantationloss are likely to have occurred. Anogenital distance was decreasedat birth in the male offspring at 500 and 750 mg/kg/day. Theepididymis was absent or underdeveloped in 9, 50, and 71% ofadult offspring (100 days old) at 250, 500, and 750 mg/kg/day,respectively, and was associated with testicular atrophy andwidespread germ cell loss. Hypospadias occurred in 3, 21, and43% of males and ectopic or absent testes in 3,6, and 29% ofmales at 250, 500, and 750 mg/kg/day, respectively. Absenceof prostate gland and seminal vesicles as well as small testesand seminal vesicles were noted at 500 and 750 mg/kg/day. Vaginalopening and estrous cyclicity, both estrogen-dependent events,were not affected in the female offspring, although low incidencesof reproductive tract malformations were observed at 500 and750 mg/kg/day. In the male offspring, DBP produced the samespectrum of effects elicited by the antiandrogen flutamide.Thus, DBP specifically impaired the androgen-dependent developmentof the male reproductive tract, suggesting that DBP is not estrogenicbut antiandrogenic in the rat at these high dose levels. Forhuman risk assessment, determining if this toxicity is metabolite-mediatedwill be critical, since marked species differences in metabolismexist.     

Subchronic, reproductive, and developmental toxicity of a fluorotelomer-based urethane polymeric product

A commercial fluorotelomer-based urethane polymeric dispersion, consisting of polymer, surfactant, and water, was evaluated in subchronic, reproduction, and developmental toxicity studies. The dispersion was administered daily by gavage to rats at dosages of 0, 50, 250, or 1000 mg polymer/kg/day or with 70 mg/kg/day of the sulfonate surfactant. Dose levels of 0, 50, 250, or 1000 mg polymer/kg/day were also used for the reproductive and developmental studies. Nasal olfactory epithelial degeneration and necrosis occurred in all dose groups in the 90-day study. Nasal adhesions were observed only in rats administered surfactant alone. Liver-enzyme alterations at 250 and 1000 mg/kg were considered to be potentially adverse effects. The subchronic no-observed-adverse-effects level (NOAEL) was 50 mg/kg. For the reproduction study, rats were dosed for 10 weeks prior to cohabitation and throughout mating, gestation, and lactation. There were no effects on reproductive function in males or females at any dosage. Thyroid weight was decreased in the 250 and 1000 mg/kg day F(1) groups unaccompanied by microscopic effects. In the developmental toxicity study, female rats were dosed from gestation days 6-20; there was no test-substance-related embryolethality, nor was there any dose-related increase in either fetal malformations. Fetal weight was minimally decreased at 1000 mg/kg/day in the presence of slight maternal toxicity; the NOAEL for developmental parameters was 250 mg/kg/day. The polymeric product was not a specific developmental or reproductive toxin.     

Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate

Adult male rats previously exposed on gestation days (GD) 12-21 to di(n-butyl) phthalate (DBP) have reproductive tract malformations, particularly agenesis of the epididymis, decreased sperm production, and Leydig cell hyperplasia and adenomas. Although similar effects are produced by the potent androgen receptor (AR) antagonist flutamide and are indicative of disruption of male sexual differentiation via an antiandrogenic mechanism, DBP is not an AR antagonist. The purpose of the study was to determine whether DBP causes pathologic changes and alterations in androgen status in the testis during the prenatal period of male reproductive tract differentiation. Pregnant CD rats were given corn oil, DBP (500 mg/kg/day), or flutamide (100 mg/kg/day) p.o. on GD 12-21. At GD 16-21, DBP caused hyperplasia of Leydig cells, many of which were 3beta-hydroxysteroid dehydrogenase- and/or AR-positive. Focal areas of hyperplasia had increased numbers of Leydig cells positive for proliferating cell nuclear antigen (PCNA). At GD 21, testis atrophy was apparent, seminiferous cords in DBP-exposed fetuses were enlarged and contained multinucleated gonocytes that, unlike controls, were PCNA-positive. DBP, but not flutamide, markedly decreased testicular testosterone levels at GD 18 and 21. Fewer epididymal ducts and reduced AR staining in some ducts were evident with DBP treatment, whereas decreased overall AR staining was seen with flutamide in the presence of mild Leydig cell hyperplasia. Leydig cell proliferation is likely a compensatory mechanism to increase testicular steroidogenesis triggered by testosterone insufficiency. The overall decrease in androgen concentration is not corrected and results in reproductive tract malformations. The multinuclearity and proliferation of gonocytes suggests an underlying Sertoli cell dysfunction.     

Reproductive and developmental toxicity of inhaled 2,3-dichloro-1,3-butadiene in rats

Inhalation developmental and reproductive toxicity studies were conducted with 2,3-dichloro-1,3-butadiene (DCBD), a monomer used in the production of synthetic rubber. In the reproductive toxicity study, Crl:CD^®(SD)IGS BR rats (24/sex/group) were exposed whole body by inhalation to 0, 1, 5, or 50 ppm DCBD (6 h/day) for approximately 10–11 weeks total, through premating (8 weeks; 5 days/week), cohabitation of mating pairs (up to 2 weeks, 7 days/week), post-cohabitation for males (7 days) and from conception to implantation (gestation days 0–7 [GD 0–7]), followed by a recovery period (GD 8–21) for presumed pregnant females. Estrous cyclicity was evaluated during premating (last 3 weeks) and cohabitation. Reproductive organs and potential target organs, sperm parameters, and GD 21 fetuses (viability, weight, external alterations) were evaluated. In the developmental study, pregnant Crl:CD^®(SD)IGS BR rats (22/group) were exposed whole body by inhalation to 0, 1, 10, or 50 ppm DCBD (6 h/day) on GD 6–20; dams were necropsied on GD 21 (gross post-mortem only) and fetuses were evaluated (viability, weight, and external, visceral and skeletal exams). During the in-life portion of the studies, body weight, food consumption, and clinical observation data were collected. At 50 ppm, gasping and labored breathing occurred in both studies during the first few exposures; body weight and food consumption parameters were affected in parental animals from both studies, but were more severely affected in the developmental study. Fetal weight was decreased in the developmental study at 50 ppm. Degeneration of the nasal olfactory epithelium was observed in the reproduction study at 50 ppm. There were no effects on reproductive function, embryo–fetal viability, or increases in fetal structural alterations in either study. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 50 ppm. The NOAEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats. The NOAEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced fetal weight at 50 ppm in the developmental toxicity study.     

Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK

Background  

Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa β ligand /receptor activator of NF-kappa β (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK.     

The role of hydrostatic pressure in foam cell formation upon exposure of macrophages to LDL and oxidized LDL

Hypertension is a major, established risk factor for atherosclerosis. How it interacts to exacerbate the cellular processes involved in atherogenesis is unclear. This initial, preliminary study examined how hydrostatic pressure influenced the formation of foam cells from human macrophages exposed to low-density lipoprotein (LDL) or oxidized LDL (OxLDL). The results demonstrated that both LDL and OxLDL, at physiological concentration, were taken up by cultured human macrophages and foam cells were formed. This led to cell detachment and death within 24h. These effects were more rapid and more pronounced in pressurized cultures. We conclude that exposure of cell cultures to cyclical hydrostatic pressure (CHP) aggravated the adverse effects of the lipids on the macrophages.     

Perfluorooctanoate: Placental and lactational transport pharmacokinetics in rats

This study was conducted to develop a quantitative understanding of the potential for gestational and lactational transfer of perfluorooctanoate (PFOA) in the rat. Time-mated female rats were dosed by oral gavage once daily at concentrations of 3, 10, or 30 mg/kg/day of the ammonium salt of PFOA (APFO) starting on gestation (G) day 4 and continuing until sacrifice. On days 10, 15, and 21G, five rats per dose level were sacrificed and blood samples were collected 2h post-dose. Embryos were collected on day 10G, amniotic fluid, placentas, and embryos/fetuses were collected on days 15 and 21G, and fetal blood samples were collected on day 21G. Five rats per dose level were allowed to deliver and nurse their litters, and on days 3, 7, 14, and 21 post-partum (PP) milk and blood samples of maternal and pup were collected 2h post-dose. All samples were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) for PFOA concentration. Concentrations of PFOA in maternal plasma and milk attained steady state during the sampling interval. The steady-state concentrations in maternal plasma were 10-15, 25-30, and 60-75 microg/mL in rats receiving 3, 10, and 30 mg/kg, respectively. Steady-state concentrations in milk were approximately 10 times less than those in maternal plasma. The concentration of PFOA in fetal plasma on day 21G was approximately half the steady-state concentration in maternal plasma. The milk concentrations appeared to be generally comparable to the concentrations in pup plasma. Pup plasma concentrations decreased from day 3PP to day 7PP, and were similar on days 7, 14, and 21PP at all dose levels. PFOA was detected in placenta (days 15 and 21G), amniotic fluid (days 15 and 21G), embryo (days 10 and 15G), and fetus (day 21G). These pharmacokinetics allow estimation of the dose to developing and nursing rat offspring following maternal exposure.     

Evaluation of the developmental toxicity of 8-2 telomer B alcohol

The potential maternal and developmental toxicity of 8-2 Telomer B Alcohol was assessed in rats. Groups of 22 time-mated female Crl:CD (SD)IGS BR rats were administered oral gavage doses as suspensions of 8-2 Telomer B Alcohol in aqueous 0.5% methylcellulose from day 6 through 20 of gestation (G) at daily doses of either 0, 50, 200, or 500 mg/kg. Under the conditions of this study, adverse maternal toxicity was produced at 500 mg kg(- 1) day(- 1) and consisted of maternal mortality, decreased body weights and body weight gains, and increased clinical observations of toxicity. One litter at 500 mg kg(- 1) day(- 1) consisted of one early resorption and was believed to be secondary to overt maternal toxicity, although single conceptus litters occur historically in this strain of rats. Developmental toxicity at 500 mg kg(- 1) day(- 1) consisted of increased fetal skeletal variations (delayed pelvic bone ossification and wavy ribs). At 200 and 500 mg kg(- 1) day(- 1), there were transient reductions in maternal feed consumption. In addition, there were slight increases in the incidence of delayed fetal skull bone ossification at 200 and 500 mg kg(- 1) day(- 1). The no-observed-adverse-effect level (NOAEL), defined as the highest dose at which adverse effects attributable to the test substance were not detected, for both maternal and developmental toxicity, is considered to be 200 mg kg(- 1) day(- 1). Thus, 8-2 Telomer B Alcohol is not considered to be a selective developmental toxicant in rats. The transient and quantitative nature of the observations in the 200 mg/kg group supports the conclusion that these findings were not adverse.     

Novel mutations in the 1alpha-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages.

Pseudovitamin D-defiency rickets (PDDR) is an autosomal recessive disorder characterized by hypocalcemia, rickets (which are resistant to treatment with vitamin D), and low or undetectable serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). The symptoms are corrected with 1,25(OH)2D treatment, and the disease is now believed to result from a defect in the cytochrome P450 component (P450c1; CYP27B1) of the renal 25-hydroxyvitamin D-1alpha-hydroxylase (1-OHase). We have studied genomic DNA from three families with PDDR and have identified the same homozygous mutation in the P450c1 gene in two of the index cases, causing a frameshift in exon 8, resulting in a premature stop codon in the heme-binding domain. The two cases in the third kindred were compound heterozygotes with missense mutations in exons 6 and 9. We have also identified a C/T polymorphism in intron 6 of the P450c1 genomic DNA. Interferon gamma-inducible 1-OHase activity in blood-derived macrophages was shown by 1,25(OH)2D synthesis in all control cells tested (37-184 fmol/h/106 cells) and those from the PDDR family parents (34-116 fmol/h/106 cells) but was totally absent from the patients' cells, indicating a defect in their macrophage 1-OHase, similar to the presumed renal defect. The assumption of similarity between the renal and macrophage P450c1 was supported by our ability to clone a 514 bp sequence, including the heme-binding region of the macrophage P450c1 cDNA from controls, which was identical to that published for both the renal and keratinocyte P450c1 cDNAs.