Richard T. Robertson

Association between Adverse Maternal and Embryo-Fetal Effects in Norfloxacin-Treated and Food-Deprived Rabbits

Norfloxacin is a new antibiotic which caused embryo-fetal toxicity in association with maternotoxicity when given orally to rabbits at 100 mg/kg/day. The intestinal flora of rabbits is unusually sensitive to many antibiotics and it was suspected that the maternotoxicity and embryo-fetal toxicity caused by oral norfloxacin were secondary to an effect on the intestinal flora. To test this idea, a teratologic study was conducted in which rabbits were dosed on Days 6 to 18 of gestation with norfloxacin given orally at 100 mg/kg/day or subcutaneously at 20 mg/kg/day. The oral treatment caused decreased food consumption (to less than 15 g/day in some animals), body weight loss, an increased resorption rate, and decreased fetal weight. Among the females in the orally dosed group, there was a significant correlation (p less than or equal to 0.005) between the effects on maternal body weight and the resorption rate. The subcutaneous treatment caused little intestinal exposure (biliary excretion = only 2-4% of dose) and no maternotoxicity or embryo-fetal toxicity, even though blood levels of drug were at least as high as those in the oral group. Since the maternotoxicity and embryo-fetal toxicity were specific to the oral route and not correlated with the level of systemic exposure, the maternotoxicity may have been secondary to an effect on the intestinal flora and the embryo-fetal toxicity may have been secondary to the maternotoxicity. The decreased food consumption observed in the oral group may have contributed to the embryo-fetal toxicity since, in a separate study, it was found that lowering the amount of food provided to rabbits on Days 6 to 18 of gestation from 150 g/day to 50 or 15 g/day also caused adverse maternal and fetal effects including, at 15 g/day, fetal malformations.

Effects of seminal vesicle and coagulating gland ablation on fertility in rats

In rats the secretions of the seminal vesicles and coagulating glands clot after ejaculation in the female tract to form a copulatory plug. The plug plays a critical role in transcervical sperm transport. Factors that influence the secretions of accessory sex glands have the potential to alter fertility without adverse effects on the gametes themselves. In the present study, the effect of surgical removal of the rat seminal vesicles and coagulating glands (SVx) on mating and fertility was investigated. SVx males were completely infertile by natural mating. Observed mating of SVx males with LHRH synchronized females confirmed that mating performance was normal and that they were capable of ejaculating sperm but could not produce copulatory plugs. In the absence of the copulatory plug, no sperm was transported into the uterus. Epididymal sperm obtained from the same males were capable of fertilization after intrauterine insemination and gave comparable numbers of embryos and unfertilized oocytes as sham operated controls. These findings underscore the importance of examining the role of both males and females during insemination and that observed matings, uterine sperm counts, and intrauterine insemination are useful adjuncts to routine fertility assessment when effects on the accessory sex glands are suspected.

Decreased fertility in male rats administered the 5α-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation

Oral administration of 80 mg/kg/day of finasteride, a potent specific inhibitor of 5 alpha-reductase, to sexually mature male Sprague-Dawley rats for 24 to 38 weeks caused an approximate 30% to 40% decrease in fertility. There were no effects on mating indices or implants per pregnant female. From the mating trials, a selected group of treated males with poor reproductive performance was compared to a selected group of control males with good reproductive performance. Observed matings showed no qualitative effects on mating behavior or ejaculation. However, finasteride-treated males did not form or formed small and improperly positioned copulatory plugs, which are required in rats to transport sperm into the uterus. Intrauterine insemination of epididymal sperm from males that were nonfertile by natural mating resulted in similar numbers of embryos and unfertilized oocytes recovered from controls and finasteride-treated males, confirming that there was no effect of finasteride on the ability of sperm to fertilize. Decreased fertility of finasteride-treated males was due to failure to form copulatory plugs and is related to decreased weight of seminal vesicles and prostate, an expected pharmacologic effect. Testes weight was unaffected. Decreased fertility in male rats after finasteride administration is considered a species specific effect. The mechanism of the decrease in rats is not likely to be relevant to species that do not form copulatory plugs.

Maternotoxicity and fetotoxicity of an angiotensin-converting enzyme inhibitor, enalapril, in rabbits

When enalapril, an angiotensin-converting enzyme (ACE) inhibitor, was orally administered to inseminated rabbits at dosages of 0.1 to 30 mg/kg/day for 13 days in a range-finding study, nephrotoxicity, as measured by elevated serum urea nitrogen concentrations, occurred at 1 mg/kg/day and higher dosages and significant (p less than or equal to 0.05) increases in fetal wastage were observed at dosages as low as 3 mg/kg/day. Saline supplementation during treatment prevented this rise in urea nitrogen. Fetal wastage was significantly (p less than or equal to 0.05) increased in the absence of maternotoxicity when saline-supplemented females were treated with enalapril at 30 mg/kg/day. A developmental toxicity study of enalapril in saline-supplemented rabbits produced no evidence of teratogenicity at 3, 10, and 30 mg/kg/day. The period of sensitivity of fetuses to the toxic effects of enalapril was found to be limited to middle-to-late gestation (Gestational Days 14-27). A single oral dose of enalapril (30 mg/kg) on Day 26 of gestation resulted in 100% fetal deaths. On the basis of the work done by Broughton Pipkin et al. [1982, J. Physiol. (London) 323, 415-422] and Broughton Pipkin and Wallace (1986, Brit. J. Pharmacol. 87, 533-542), which demonstrated that the sheep fetus becomes markedly hypotensive when the dam is treated with captopril or enalapril during late pregnancy, we believe that the observed fetotoxicity of enalapril in rabbits is also due to fetal hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium loss as a causative factor for skeletal malformations in rats produced by indacrinone: A new investigational loop diuretic

Indacrinone (MK-196), an investigational loop diuretic, was administered to female CRCD rats from Days 6 to 17 of gestation at doses of 40, 80, or 120 mg/kg/day. Dose-related increases in wavy ribs and malformations of the scapula and humerus were apparent in fetuses from all drug-treated groups. Studies on the individual enantiomers of MK-196 showed that the teratogenic potency was associated with the relative saluretic activity of the enantiomers. Furosemide, a widely used diuretic with pharmacologic activity similar to that of MK-196, was administered to rats on Days 6 to 17 at doses of 37.5, 75, 150, or 300 mg/kg b.i.d. Dose-related increases in wavy ribs occurred in all drug treatment groups. At 150 mg/kg b.i.d. there were malformations in the scapula and humerus identical to those described for MK-196. Providing a 1% solution of KCl during the dosing period reduced the incidence of wavy ribs in fetuses from MK-196-treated dams by 90% and eliminated the scapular and humeral malformations. Coadministration of amiloride was also found to antagonize the teratogenicity of MK-196. The results indicate that high doses of diuretics with similar pharmacologic activities can produce identical skeletal malformations in rats, and that this teratogenicity is related to hypokalemia.

Reversible decreases of fertility in male Sprague-Dawley rats treated orally with finasteride, a 5α-reductase inhibitor

Finasteride, a 5 alpha-reductase inhibitor, was investigated for its effects on fertility in male rats as part of its preclinical safety assessment. Studies were initiated when the male Sprague-Dawley rats were either young (4 to 6 weeks old) or mature (15 weeks old). Treatment duration ranged from 6 to 32 weeks. Each male was cohabited with two untreated females at various periods during and after treatment. Litter parameters were evaluated on either day 14 or 20 of gestation. Males were necropsied at the end of treatment or 7 to 11 weeks following the end of treatment. The major findings of these studies were that 1) young rats given 20 to 80 mg/kg/day of finasteride first showed mild to moderate decreases in fertility after 12 weeks of treatment, whereas mature males (given only 80 mg/kg/day) did not show a similar decrease until 24 weeks of treatment, 2) fewer copulatory plugs and atrophy of prostates and seminal vesicles were associated with finasteride treatment, 3) the decreased fertility was only partial (ie, fertility index did not decrease below 48% of control in any study) and was not due to decreases in mating, 4) formation of copulatory plugs, organ weights, and fertility returned to normal levels after at least 6 weeks of drug withdrawal, and 5) the testes showed no histologic or weight changes that would explain the effect on fertility. These results show that the decreased fertility in male rats was associated with finasteride-induced inhibition of accessory gland secretions, an expected pharmacologic effect.

Exposure of Rats to Lead Nitrate in utero or Postpartum; Effects on Morphology and Behavior

Female rats were administered lead (Pb), as the nitrate salt, on day 17 of pregnancy (5 or 25 mg/kg i.v.) or throughout lactation (5 or 25 mg/kg/day p.o.). There were adverse effects on weights of females receiving Pb on day 17. At 25 mg/kg i.v. gestation was significantly prolonged. In both groups treated intravenously, average pup weight on day 1 postpartum was significantly reduced and there was a significantly higher mortality than in controls, and hydrocephalus occurred. Survival rate and weight gain of pups from dams that received Pb throughout lactation was not different from controls. Brain weights and histomorphology of all groups was normal. Behavior in male offspring, as measured by open-field activity, rotorod or passive avoidance tests, was unaffected by exposure to Pb.

Evaluation of the Teratogenicity and Pharmacokinetics of Diflunisal in Cynomolgus Monkeys

This study examined the pharmacokinetics and potential teratogenicity of the nonsteroidal antiinflammatory drug, diflunisal, in cynomolgus monkeys. Pregnant cynomolgus monkeys were administered 0.5% methyl cellulose, 20 mg/kg/day diflunisal, or 80 mg/kg/day diflunisal on Days 25 to 48 of gestation. There was no evidence of maternal toxicity, increased abortion rate, fetal growth retardation, or malformation. These data demonstrate that diflunisal is not teratogenic in cynomolgus monkeys over a dosage range of 20 to 80 mg/kg/day. Peak plasma levels of diflunisal were found 1 hr after oral administration of [14C]diflunisal at a dosage of 60 mg/kg and declined to low levels by 24 hr. The plasma elimination half-life was calculated to be 10.2 hr over the period of 1 to 8 hr postadministration. Intact diflunisal accounted for 96.4% of total plasma radioactivity at 0.5 hr and declined to a value of 74% at 8 hr. Plasma protein binding averaged greater than 99% over a concentration range of 62.5 to 250 micrograms/ml. Urinary excretion of diflunisal and metabolites averaged 66.5% of the dosage over the first 4 days postadministration, compared with 0.8% in the feces. The majority of activity represented conjugates of diflunisal. Embryo concentrations of diflunisal on Days 35 to 37 of gestation were 0.7 and 1.1% of maternal plasma level at 4 hr postadministration of 20 or 60 mg/kg, respectively.

The Rapid and Sensitive Detection of Perturbations in Spermatogenesis: Assessment by Quantitative Dual Parameter (DNA/RNA) Flow Cytometry

This presentation will concentrate on the emerging field of flow cytometry. The first portion will be a review of the state-of-the-art applications of flow cytometry in the field of biomedical research; the second portion will describe the results of a preliminary study using a published technique that is useful in detecting cellular perturbations in germ cells. The model used in this study was the testis from the Sprague-Dawley rat. Adult rats received a single bolus of busulfan, and their testes were examined up to 56 days postadministration.