Carole A. Kimmel

A Rapid Procedure for Routine Double Staining of Cartilage and Bone in Fetal and Adult Animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.

The Developmental Toxicity of Bisphenol A in Rats and Mice

Bisphenol A (BPA) was evaluated for developmental toxicity in CD rats (0, 160, 320, or 640 mg/kg/day) and CD-1 mice (0, 500, 750, 1000, or 1250 mg/kg/day) dosed daily by gastric intubation on Gestational Days 6 through 15. Timed-pregnant dams were sacrificed 1 day prior to parturition, the uterine contents were examined, and all fetuses were examined for external, visceral, and skeletal malformations. In rats, maternal weight gain during gestation, weight gain corrected for gravid uterine weight, and weight gain during treatment were significantly reduced at all BPA doses. Gravid uterine weight and average fetal body weight per litter were not affected by BPA. No increase in percentage resorptions per litter or percentage fetuses malformed per litter was detected. In mice, maternal mortality occurred at all BPA doses, reaching 18% at the high dose, which also produced a significant decrease in maternal body weight gain during gestation and treatment. Weight gain corrected for gravid uterine weight was not affected by BPA. Reductions in gravid uterine weight and average fetal body weight were observed with the 1250 mg/kg dose of BPA. Relative maternal liver weight was increased at all doses of BPA. There was a significant increase in the percentage of resorptions per litter with 1250 mg BPA/kg/day. Malformation incidence was not altered by BPA. Thus, BPA treatment at maternally toxic dose levels during organogenesis produced fetal toxicity in mice but not in rats and did not alter fetal morphologic development in either species.

The developmental toxicity of ethylene glycol in rats and mice

Timed-pregnant CD rats and CD-1 mice were dosed by gavage with ethylene glycol (EG) in distilled water on gestational days (gd) 6 through 15 (0, 1250, 2500, or 5000 mg kg-1 day-1 for rats; and 0, 750, 1500, or 3000 mg kg-1 day-1 for mice). Females were observed daily during treatment, but no maternal deaths or distinctive clinical signs were noted. Dose-related decreases in maternal weight gain during treatment were significant at all doses in rats and at the mid and high doses in mice. Gravid uterine weight was reduced in both species at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. At termination (gd 20, rats; gd 17, mice), the status of uterine implantation sites was recorded, and live fetuses were weighed and examined for external, visceral, and skeletal malformations. Dose-related increases in postimplantation loss per litter were observed in both species with the high dose significantly above controls only in rats. Fetal body weight per litter was significantly reduced at the mid and high doses in rats and at all doses in mice. The percentage of malformed live fetuses per litter and/or the percentage of litters with malformed fetuses was significantly elevated in all EG dose groups and greater than 95% of litters were affected at the high dose for each species. A wide variety of malformations were observed; the most common in both species were craniofacial and neural tube closure defects and axial skeletal dysplasia. EG produced severe developmental toxicity in two rodent species at doses that apparently failed to produce any serious maternal effects.

Blood flow changes and conceptal development in pregnant rats in response to caffeine

Alterations in blood flow to the uterus and its contents during pregnancy have been suggested to account for the teratogenicity and/or embryotoxicity of several agents, including caffeine. Using a radioactive microsphere technique, blood flow to several maternal organs, including ovary, uterus, decidua, and chorioallantoic placenta (CAP), was measured following a single dose of 0 or 120 mg/kg caffeine by gavage to pregnant CD rats on Day 12 of gestation. At 1 or 4 hr after treatment, animals were anesthetized and strontium 85-labeled microspheres (25 micrometers diam) were infused into the left ventricle. Whole body and tissue radioactivity were determined. Maternal cardiac output (CO) and absolute flow (f1; ml/min), relative flow (f2; ml/min/g tissue), and flow as %CO (f3) to each tissue were calculated. Maternal CO was not altered. All blood flow parameters for ovaries, uterus, and bladder were reduced in treated animals at both time points except for absolute flow (f1) to the ovaries at 1 hr. Decidual changes included reduced weight at 1 hr, reduced f2 at 4 hr, and reduced f1 and f3 at both times. However, CAP weight and blood flow wre not significantly altered by caffeine treatment. Examination of conceptuses from thse litters, and from other animals at 24 hr after treatment or at term did not reveal any significant effect of this dose of caffeine on viability, growth, or physical development. The ratio of embryo to maternal blood caffeine concentrations was approximately 1, indicating free transfer of caffeine to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of maternal diet in the developmental toxicology of ethanol

A commonly observed result of ethanol administration to experimental animals is a reduction in voluntary dietary intake. The purpose of this investigation was to examine the role of this reduction in the developmental/reproductive toxicity of ethanol. To achieve this objective, female C3H mice were administered one of the following diets from Day 0 to Day 17 of pregnancy: (1) liquid diet, (2) liquid diet plus 4.1% w/v ethanol, (3) an amount of liquid diet equal to that consumed by the liquid diet plus 4.1% w/v ethanol group, i.e., pair-fed, (4) liquid diet fortified with protein, fats, carbohydrates, vitamins, and minerals, or (5) fortified diet plus 4.1% w/v ethanol. An important aspect of this experiment was the 2 X 2 factorial design, which permitted the analysis of the effects of ethanol consumption, diet intake reduction, and potential interactions between these two factors. After maternal death on Day 17 of gestation, fetuses were examined for evidence of alterations in growth and/or development. Analysis of the results revealed that decreased dietary intake, but not ethanol consumption resulted in a decrease in fertility, maternal pregnancy weight gain, and litter size. In contrast, both decreased dietary intake and ethanol consumption significantly inhibited fetal growth and development. However, no interaction between these two factors was demonstrated, indicating that ethanols fetotoxic effects occurred, regardless of maternal dietary intake. We conclude that, depending on the endpoint examined, maternal nutrition plays a key role in experimental ethanol fetotoxicity. However, this agent is fetotoxic, even in the well-nourished animal.

Postnatal toxicity following prenatal reserpine exposure in rats: Effects of dose and dosing schedule

Pregnant CD rats were treated subcutaneously with 0, 0.1, 0.33, or 1.0 mg reserpine/kg/day either on Days 12-15 or on Days 16-19 of gestation. Dams were allowed to deliver and litters (4 +/- 1 of each sex) were weighed weekly and held to 21 days of age. Basal ornithine decarboxylase (ODC) activity and neurochemical determinations were made on hearts and brains, respectively, from pups culled from litters on postnatal Day 1, and from two males and two females/litter at 21 days of age. Following both treatment schedules, the high dose of reserpine resulted in maternal weight loss during dosing, increased stillborn pups, reduced pup weight at birth, retarded postnatal growth, and decreased survival to 21 days of age. Basal cardiac ODC activity was reduced to 33% of control levels only on Postnatal Day 1 in both high-dose groups, while absolute heart weight decreased and relative heart weight increased in these pups. Whole-brain concentrations of two neurotransmitter metabolites, 3-4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), were increased only at Postnatal Day 1 in the high dose group treated on Days 12-15 of gestation. No other changes were found in concentrations of these metabolites or in the transmitters dopamine and serotonin. The only effect found following administration of 0.33 mg/kg reserpine was a reduction in maternal weight gained during both dosing periods. No signs of toxicity were observed following low-dose exposure on either schedule. Most previously reported postnatal functional studies following reserpine exposure have used mid- to late-gestational treatment with 1.0 mg/kg, a dose shown here to result in marked overt maternal and fetal toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

The Developmental Toxicity of Orally Administered Oxytetracycline in Rats and Mice

Timed-pregnant CD rats and CD-1 mice were dosed by gavage with oxytetracycline hydrochloride (OXT) in corn oil on gestational days (gd) 6-15 (0, 1200, 1350, or 1500 mg/kg/day for rats; 0, 1325, 1670, or 2100 mg/kg/day for mice). Deaths among treated females occurred in a dose-related manner in all OXT dose groups (2-7%, mice; 5-24%, rats), but no maternal deaths occurred in the vehicle control groups. Significant dose-related decreases in maternal weight gain during treatment, as well as for corrected gestational weight gain (i.e., maternal gestational weight gain minus gravid uterine weight), were observed at all doses in rats but not in mice. Gravid uterine weight was reduced in a dose-related manner only in mice, with the high-dose group significantly reduced compared to the control group. At termination (gd 20, rats; gd 17, mice), the status of uterine implantation sites was recorded and live fetuses were weighed. Fetuses were examined for external, visceral, and skeletal abnormalities. There were no significant effects of OXT in either species on the incidence of postimplantation loss (resorptions plus dead fetuses) or malformations. In both species, there was a significant trend toward reduced fetal body weight, and each group of rats receiving OXT was significantly reduced compared to the control group. Administration of OXT during organogenesis at doses exceeding the therapeutic range for humans produced maternal and fetal toxicity, but did not produce any treatment-related increase in malformations.

The evaluation of the developmental toxicity of hydrochlorothiazide in mice and rats.

Timed-pregnant CD-1 outbred albino Swiss mice and CD Sprague-Dawley rats were administered hydrochlorothiazide (HCTZ, USP) in corn oil by gavage during major organogenesis, Gestational Days (GD) 6 through 15. The doses administered were 0, 300, 1000, or 3000 mg/kg/day for mice and 0, 100, 300, or 1,000 mg/kg/day for rats. Maternal clinical status was monitored daily during treatment. At termination (GD 17, mice; GD 20, rats), confirmed pregnant females (20-27 per group, mice; 36-39 per group, rats) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In mice, no maternal mortality was observed. However, clinical signs including dehydration, piloerection, lethargy, and single-day weight loss appeared to be dose-related. HCTZ had no effect on maternal weight gain or water consumption, gravid uterine weight, relative maternal liver weight, or relative maternal kidney weight. There was no definitive evidence of embryotoxicity or fetal toxicity for mice on GD 17. Thus, the no observed adverse effect level (NOAEL) for both maternal and developmental toxicity was 3000 mg/kg/day. In rats, HCTZ had no effect on maternal survival, clinical signs, or water consumption. Clinical signs were not dose-related. Maternal weight gain during treatment was depressed at 1000 mg/kg/day. Gravid uterine weight and relative maternal liver weight were unaffected. Relative maternal kidney weight was slightly (7-8%) increased at all dose levels, but there was no evidence of a dose response. Thus, the maternal NOAEL for rats was 300 mg/kg/day, based on decreased maternal weight gain during treatment at 1000 mg/kg/day.(ABSTRACT TRUNCATED AT 250 WORDS)