Long-term evaluation of the diethylstilbestrol (DES) syndrome in adult female rhesus monkeys (Macaca mulatta).

This study was designed to examine the transplacental effects of diethylstilbestrol (DES) in rhesus monkeys (Macaca mulatta). Nineteen pregnant females received an approximate human therapeutic dose of 1 mg/day DES beginning on gestational day 21 (Group I), day 100 (Group II), or day 130 (Group III) until term. Colposcopic examination and vaginal biopsies were performed in eight female offspring at 6- and 12-month intervals beginning at 3.5 years of age until death between 5 and 12 years of age. Gross and histological evidence of vaginal adenosis was observed in five of eight (62.5%) females. Adenosis, which occurred in all treatment groups, did not develop into neoplasia; rather, it underwent metaplasia and reversion to squamous epithelium, a frequent observation in human DES cases. The vaginal ridging and/or cervical hooding characteristic of the human syndrome was observed in all exposed females. Although menstrual cyclicity was not impaired, there appeared to be a lower pregnancy rate in treated animals compared to age-matched controls. This long-term evaluation of reproductive morphology and function in the rhesus monkey has provided a useful model for studying the history of benign vaginocervical abnormalities induced by prenatal DES treatment.

Developmental toxicity and pharmacokinetics of oral and intravenous phenytoin in the rat.

Correlations between oral and intravenous (i.v.) doses of phenytoin, maternal plasma levels, and subsequent developmental toxicity were examined in the Sprague-Dawley rat. Oral administration of 150 to 1500 mg/kg and i.v. administration of 25 to 100 mg/kg phenytoin from gestational days (GD) 8 to 17 resulted in a dose-dependent increase in maternal death and toxicity [impaired motor function, decreased maternal weight gain (oral dose only)], embryolethality, and intrauterine growth retardation, in addition to significant increases in craniofacial (1125 mg/kg oral; 75 mg/kg i.v.) and urogenital (1125 mg/kg oral) malformations. Pharmacokinetic sampling in oral and i.v. groups on GD 8-9 and 16-17 revealed significant increases in maternal drug exposure over the treatment period, as evidenced by 2- to 3-fold increases in total plasma phenytoin (bound + free) half-life, area under the concentration curve, peak concentration (oral dose only), and decreases in clearance. These findings emphasize the importance of pharmacokinetics in the evaluation of phenytoin-induced developmental toxicity.

Developmental toxicity and pharmacokinetics of phenytoin in the rhesus macaque: an interspecies comparison.

The developmental toxicity and pharmacokinetic fate of phenytoin in the pregnant rhesus macaque (Macaca mulatta) were examined. Oral administration of 60 to 600 mg/kg phenytoin once daily from gestational day 21 to 50 resulted in dose-dependent maternal toxicity of the central nervous system and gastrointestinal tract and an increase in embryonic loss, but no teratogenic insult. Sustained plasma levels as high as 40 micrograms/mL of total phenytoin occurred at the beginning of the treatment period. However, significant increases in the rate of elimination resulted in the reduction of total phenytoin exposure as treatment progressed. This was evidenced by large increases in phenytoin clearance, and decreases in elimination half-life and area under the time versus plasma concentration curve. Maternal toxicity, but not embryolethality, correlated with plasma phenytoin levels. Interspecies comparisons of these parameters from published data were evaluated in the mouse, rat, rabbit, rhesus macaque, and human.

Embryonic development in Erythrocebus patas.

The developmental stages of 12 Erythrocebus patas embryos, ranging in gestational age from 30 to 50 days, is described. The pattern of embryogenesis in E. patas closely parallels the anatomic characteristics of human and other nonhuman primate embryos between stages 12 and 23. However, there is a delay in development in E. patas similar to that observed in human embryos which differs from the macaques and baboons. This temporal difference in the embryonic period is an important factor in the design and analysis of early pregnancy studies in this species.

The effects of prenatal diethylstilbestrol exposure on the genitalia of pubertal Macaca mulatta. II. Male offspring.

In order evaluate the potential embryotoxic and fetotoxic effects of in utero diethylstilbestrol (DES) exposure on the developing offspring, 19 pregnant rhesus monkeys were administered 1 mg/day DES orally beginning on either day 19 (group I), 100 (group II) or 130 (group III) or gestation and termination on the day of natural birth or cesarean section. Five of ten male offspring are alive at 7 years of age. At 4 1/2 years of age, three of these five offspring exhibited one or more abnormalities of the external genitalia, including testicular hypoplasia, preputial adhesions and undescended testes. Semen analysis following rectal electroejaculation and testicular biopsies at 5 1/2 years of age confirmed two cases of testicular hypoplasia. Semen evaluation, testicular biopsies and analysis of serum testosterone levels at 6 1/2 years of age indicated normal testicular morphology and function in all DES-exposed males as compared with colony controls. Our study, therefore, suggests that DES may affect maturation of the reproductive tract as indicated by a delay in the normal breakdown of preputial adhesions in addition to gross and microscopic evidence of testicular hypoplasia during the postpubertal period between 4 1/2 and 5 1/2 years of age. Further observations on breeding performance and fertility are required to evaluate the long-term effects of DES in this species.

Nonhuman primates and teratological research.

Nonhuman primates were first recognized as models for the study of developmental toxicity (teratology) following the thalidomide tragedy. Since that time they have played important roles in both testing of drugs for human safety and as models for studying specific malformations commonly seen in children. Although in vitro and alternative test systems using lower animal forms or simplified test systems have been incorporated into developmental toxicity studies, whole animal testing will be required for the foreseeable future because of the complex relationship of the maternal/embryofetal/placental unit. The nonhuman primate will be particularly valuable where equivocal results are experienced in other commonly used laboratory species, when the drug/chemical is likely to be used during pregnancy, and for human-derived biotechnical products which often are not bioactive in nonprimate species.

Renal concentrating ability in the uninephrectomized rat.

To investigate the effects of uninephrectomy on renal concentrating ability, studies were performed on unanesthetized rats 5-11 days after uninephrectomy (UN) or a sham operation (SO). Female rats were deprived of water for 27 h prior to the infusion of inulin and para-aminohippurate and urine collection. They were also preconditioned to being handled and to the experimental locale. During a nondiuretic state urine osmolality was the same for all UN and SO groups (mean about 1,700 micro osmol/g H2O), whereas the mean solute excretion rate (micro osmol/min per kg body wt per kidney) was 74 in the UN and 35 in the SO rats. When SO rats were infused with mannitol or isotonic saline to increase their solute excretion rate per kidney to the level of the UN rats, urine osmolality dropped 200-1,000 micro osmol/g H2O; when urea was infused, urine osmolality did not drop. Thus, after uninephrectomy and a consequent doubling of the solute excretion rate per kidney, renal concentrating ability was higher than predicted on the basis of a comparable but acute elevation of the solute excretion rate. The glomerular filtration rate was about 17 ml/min per kg body wt in the SO rats and was 1.2 times greater (on a per kidney basis) in the UN rats. These exceptionally high glomerular filtration rats are attributed to preexperimental conditioning of the rats and the absence of stress during urine collection.

Abnormal development of blastocysts and blastomeres in the rhesus monkey.

Preimplantation stages were collected from normal rhesus monkeys by flushing of the uterine lumen. There was a high incidence of abnormal morulae and blastocysts (25%), as well as abnormal cells and cell death in otherwise normal blastocysts. In addition to cell and nuclear fragmentation, clustering of organelles in the center of the blastomeres was a common feature in early degeneration of ova and blastomeres. Large isolated cells that lagged in cytological development were found both in large numbers in an abnormal blastocyst and as individual cells in normal blastocysts. One apparently abnormal blastocyst was composed of normal trophoblast and inner cell mass cells, but lacked desmosomes and was collapsed. The cytological study of development of the blastocyst indicated that assessment of viability of primate blastocysts by light microscopic observation of morulae and blastocyst stages is probably more fraught with difficulty than might appear from studies of common laboratory animals.

Evaluation of valproic acid (VPA) developmental toxicity and pharmacokinetics in Sprague-Dawley rats.

This study was undertaken to assess the pharmacokinetics and developmental toxicity of the anticonvulsant, valproic acid (VPA), a human teratogen, in Sprague-Dawley rats. Oral administration of 200-800 mg/kg VPA (5-20x human therapeutic dose) from Gestational Days (GD) 8 to 17 resulted in increasing maternal toxicity at the higher doses with 100% maternal lethality at 800 mg/kg. Although there was an increased incidence of resorptions at 600 mg/kg (48 +/- 43%) compared to controls (18 +/- 24%), it was not statistically significant. Fetal examination on GD 20 revealed dose-dependent fetal growth retardation (p less than or equal to 0.05) as evidenced by decreased fetal weight and length in addition to underossification of both the axial and appendicular skeleton. The incidence of skeletal defects, including abnormal vertebrae, ribs, and craniofacial dysmorphia, also increased with higher doses of VPA. Cardiac anomalies observed in the two highest treatment groups consisted of great vessel malformations with or without associated ventricular septal defects (VSDs). Urogenital defects were also noted in the 600 mg/kg group. The plasma elimination half-life on GD 8 was 1.0 +/- 0.3 hr at 200 mg/kg and 2.3 +/- 0.7 hr at 600 mg/kg. Maximal concentrations of total and free drug were 341 +/- 18 micrograms/ml and 181 +/- 11 micrograms/ml, respectively, in the low-dose group and 911 +/- 379 micrograms/ml and 542 +/- 224 micrograms/ml in the high-dose group. No significant changes in any pharmacokinetic parameters (t1/2, AUC, Cmax, tmax) were observed over the 10-day treatment period at either dose level.

Embryotoxicity of sex steroidal hormone combinations in nonhuman primates: I. Norethisterone acetate + ethinylestradiol and progesterone + estradiol benzoate (Macaca mulatta, Macaca fascicularis, and Papio cynocephalus).

Two sex steroid hormone combinations which have been used clinically as tests for detection of early pregnancy were examined for embryotoxic effects in macaques and baboons. Norethisterone acetate and ethinyl estradiol (NEA + EE) were orally administered to rhesus and cynomolgus monkeys and baboons at dosages ranging from one to 1,000 times the human dose equivalent (HDE) during days 20-50 of pregnancy. Progesterone and estradiol benzoate (P + EB) were delivered by two to six intramuscular injections to rhesus and cynomolgus monkeys between gestational days 20 and 35 at 0.1-25 X HDE. Fetuses were examined following cesarean section at 100 +/- 2 days (NEA + EE) or at term (P + EB). The results showed increased embryolethality over controls at 100-1,000 X HDE (NEA + EE) and at 10 and 25 X HDE (P + EB). Besides growth retardation, isolated cases of minor nongenital malformations were observed only in cynomolgus monkeys following treatment with both hormone combinations mainly at embryolethal dose levels and were considered spontaneous in nature. Virilization of female cynomolgus fetuses following NEA + EE treatment was manifested as two cases of clitoral enlargement in the 300 X HDE group and two cases of increased anogenital distance with reduced vaginal opening in the 1,000 X HDE group. The highest dose of NEA + EE was also maternally toxic, as two maternal deaths occurred at the end of the treatment period. One dead female cynomolgus fetus exposed to P + EB (10 X HDE) also exhibited masculinized external genitalia.(ABSTRACT TRUNCATED AT 250 WORDS)