Effect of dietary protein, zinc, and carbon monoxide on fetal zinc concentration in mice

Dietary protein and zinc deficiencies known to be detrimental to the developing fetus are common in pregnant women in developing countries. Everyone in modern society is at risk of exposure to carbon monoxide (CO). This study was conducted to observe the effect of dietary protein, zinc, and exposure to CO on the fetal zinc concentrations by factorial experimentation. Pregnant mice of CD-1 strain were maintained on 17% (control) or 9% (deficient) protein diets mixed with deficient, normal (control), or supplemental zinc throughout gestation. The dams in each dietary group were exposed to air (control) or 500 ppm CO in air in environmental chambers from gestation day 8 to gestational day 18. The dams were sacrificed on d 18 and fetal zinc levels were measured by atomic absorption spectrophotometry. Carbon monoxide levels used in this study had no significant effect on fetal zinc concentration in any treatment group. When both dietary protein and zinc levels were normal, the mean fetal zinc concentrations were higher than all other dietary protein/zinc combinations (15.2±6.0 and 14.2±4.1 μg Zn/g of tissue for 0 and 500 ppm CO levels). However, when dietary protein levels were deficient, supplemental zinc increased the fetal zinc concentrations significantly (12.7±3.8 and 13.1±0.3.6 μg Zn/g of tissue, in 0 and 500 ppm CO groups) as compared to zinc-deficient groups (8.7±3.0 and 10.0±3.3 μg Zn/g of tissue in 0 and 500 ppm CO groups). The results of this study may be relevant to populations that experience both marginal zinc and protein diets during gestation.     

Interaction of maternal protein and carbon monoxide on pup mortality in mice: implications for global infant mortality

BACKGROUND: The United States Surgeon General declared 2005 as the "Year of Healthy Child." To improve the health of all children, we need to start before pregnancy, with their mothers. Unfortunately, protein deficiency in the diets of poor pregnant mothers in developing countries is widespread. Carbon monoxide (CO) pollution is serious public health problem in developed and developing countries. METHODS: A two-way factorial experimental design was used. Mice were maintained on 27%, 16%, 8%, or 4% protein diets. Dams were exposed to 0 ppm (control), 65 ppm, or 125 ppm CO in air, in environmental chambers for 6 hr/day during the first 2 weeks of pregnancy. Controls were also subjected to environmental chamber conditions. Food and water were available at all times. Animals were allowed to deliver, and data on pup mortality was recorded. RESULTS: Litter size was not affected by CO exposure, but was directly related to the dietary protein levels. Pup weight was inversely related to the CO exposure level, and directly related to the dietary protein levels. Pup mortality on date of birth was increased by CO exposure and was inversely related to the dietary protein levels. Pup mortality at 1 week of age was increased by CO exposure and 55% of all pups died in 125 ppm CO exposed group. Pup mortality at 1 week of age was inversely related to dietary protein levels. All pups in the 4% dietary protein and in all concentrations of CO died. All pups in the 8% protein group and in all CO concentrations died except in 125 ppm CO group. Pup mortality in the 16% dietary protein group ranged from 14.8% in 0 ppm to 36.8% in 65 ppm CO groups. Pup mortality in the 27% dietary protein group ranged from 14.3% in the 0 ppm to 41.1% in the 125 ppm CO groups. CONCLUSIONS: DATA suggest that protein deficiency and CO exposure enhance pup mortality. The protein and CO also interact to increase pup mortality in 16% and 27% protein groups. Carbon monoxide exposure, along with protein deficiency during gestation, may be contributing factors for high rates of infant mortality in developing countries. The results of the study also suggest that un-vented combustion for heating and cooking, ambient pollution, and biomass smoke may have a major impact on the health of children worldwide; and may explain the causes of high infant mortality in poor countries and some sections of the United States population.     

Evaluation of heart malformations in B6C3F1 mouse fetuses induced by in utero exposure to methyl chloride

C57BL/6 female mice impregnated by C3H males mice to produce B6C3F1 fetuses were exposed daily for six hr to atmospheres containing 0, 250, 500, or 750 ppm methyl chloride, from gestation day 6 to gestation day 18. There were 74 to 77 females with copulation plugs per exposure concentration. Females exposed to 750 ppm ethyl chloride exhibited ataxia commencing on the seventh day of exposure (gestation day 12). They also showed hypersensitivity to touch or sound, tremors and convulsions. Six females in the 750 ppm group died and one was euthanized in extremis prior to scheduled sacrifice. On gestation day 18, all other females were euthanized for evaluation. Only dams exposed to 750 ppm exhibited significant decrease in body weight by gestation day 18, weight gain during the gestation period, and absolute weight gain (weight gain minus gravid uterine weight) versus controls. There were no treatment related-effects on these parameters in the other exposure groups. None of the groups exhibited exposure-related differences in pregnancy rate, gravid uterine weight, or maternal liver weight. There were no differences in the numbers of implantations, resorption, dead fetuses, nonlive (dead plus resorbed) fetuses, live fetuses, sex-ratio, or mean fetal body weight per litter. There was a significant exposure-related increase in the number and percentage of affected (nonlive plus malformed) fetuses per litter with the incidence of affected fetuses in the 750 ppm group significantly higher than controls. There was a statistically significant increase in the incidence of heart defects in the 500 and 750 ppm group relative to controls. Of the 37 fetuses in the study with heart defects, 23 were females, 14 were males. The heart defects observed included: absent or abnormal tricuspid valve, reduced number of papillary muscles and/or chordae tendineae on the right side, small right ventricle, globular heart, and white spots in the left ventricular wall. Multiple malformations were observed in one fetus from the 500 ppm group and in three fetuses in the 750 ppm group. It is concluded that methyl chloride inhalation exposure to pregnant C57BL/6 mice from gestation day 6 through gestation day 17 resulted in maternal toxicity only at the 750 ppm exposure concentration and was teratogenic to B6C3F1 conceptuses at exposure concentrations of 750 and 500 ppm, leading to fetal heart malformations. No evidence of embryo or fetotoxicity other than teratogenicity was seen at any of the exposure concentrations employed. No maternal, embryo or fetotoxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 250 ppm of methyl chloride.     

Zinc supplementation to protein-deficient diet in CO-exposed mice decreased fetal mortality and malformtion

In developing countries, diet during pregnancy is frequently low in both protein and zinc contents and exposure to CO is common because of environmental pollution and smoking. This study was conducted to evaluate whether zinc supplementation ameliorates fetal mortality and malformations in protein-deficient, CO-exposed mice. Pregnant mice of the CD-1 strain were maintained on 17% (reference) or 9% protein diets mixed with deficient, normal, or supplemental zinc throughout gestation. The dams in each dietary group were exposed to air (control) or 500 ppm CO in air in environmental chambers from gestation days 7–18. As compared to the control group (normal protein, normal zinc), the incidence of fetal mortality was 66.8% and 57.2% higher, respectively, and malformation incidence was 74.4% and 72.4% higher (0 and 500 ppm CO, respectively) in mice fed both deficient protein-zinc diets. However, the highest malformation rate was observed in the group with normal protein, deficient zinc (96% mortality in both 500 and 0 ppm CO, as compared to the reference group, p<0.0001). The fetal mortality rate was −3.5% (0 ppm CO) and 25.4% (500 ppm CO) lower in zinc-supplemented, protein-deficient groups compared to the control group. There was a significant negative association between fetal zinc concentrations and fetal malformations (p≤0.001). The result of this study might be relevant to populations that are exposed to CO and or consume marginal zinc and protein diets during gestation.     

Gastroschisis is caused by the combination of carbon monoxide and protein-zinc deficiencies in mice

BACKGROUND: Gastroschisis is a rare congenital defect of the abdominal wall. Its occurrence is noted primarily in the offspring of young mothers who often smoke during pregnancy. The incidence of gastroschisis has been increasing in many countries in recent years. The etiology of gastroschisis is not known. METHODS: Pregnant mice of CD‐1 strain were maintained on 17 and 9% protein diets mixed with deficient, normal, or supplemental zinc levels throughout gestation. The dams in each protein‐zinc diet group were randomly divided in two groups. One group was exposed to air (control) and the other to 500 ppm carbon monoxide (CO) in air, in environmental chambers, from gestation days (GD) 8–18. The dams were sacrificed by carbon dioxide asphyxiation on GD 18, and data on malformations was collected. RESULTS: The rates of fetal mortality and malformations were increased by protein and zinc deficiencies. Carbon monoxide exposure also increased fetal mortality. In the low protein group, the rate of fetal mortality was inversely related to the dietary zinc level, and the rate of fetal malformations was highest in the zinc deficient group. The incidence of gastroschisis in the low protein/zinc deficient/CO exposed group was 47%, and 60% of the litters were affected. The incidence of gastroschisis in the rest of the low protein/zinc diets/air or CO groups was 0. CONCLUSION: The data indicates that gastroschisis is caused by the combination of protein‐zinc deficiencies and carbon monoxide exposure during gestation. The finding may be relevant to human populations that experience protein and zinc deficiencies during gestation, and are exposed to CO pollution, or cigarette, or marijuana smoke during pregnancy. Birth Defects Res B 68:355–362, 2003. © 2003 Wiley‐Liss, Inc.     

Prenatal exposure to low levels of carbon monoxide alters sciatic nerve myelination in rat offspring

Prenatal exposure to low concentrations of carbon monoxide (CO, 75 and 150 ppm from day 0 to day 20 of gestation), resulting in maternal blood HbCO concentrations equivalent to those maintained by human cigarette smokers, leads to subtle myelin alterations in the sciatic nerve of male rat offspring. The rapid growth spurt in pup body weight was related to the period of maximal increase in myelin sheath thickness in both control and CO-exposed animals. A significant reduction in myelin sheath thickness of sciatic nerve fibers, paralleled by changes in the frequency distribution, occurred in both 40- and 90-day-old rats exposed in utero to CO (75 and 150 ppm). Myelin deficit observed in 75 and 150 ppm CO-exposed animals showed up only after the major spurt in myelination but not early during development. The subtle myelin alterations observed in CO-exposed offspring were not accompanied by changes in developmental pattern of axon diameters and did not result in a gross impairment of motor activity. These results suggest that the myelination process is selectively targeted by a prenatal exposure model simulating the CO exposure observed in human cigarette smokers.     

Light and electron microscopic study of fetal lung following maternal exposure to methylmercuric chloride

Varying dose levels of methylmercuric chloride (MMC), 1000 ppm (5 mg through 15 mg/kg of body weight), were administered via an intragastric tube to pregnant ICR Swiss/Webster mice on day 9 of gestation. The animals were killed on gestational day 18 and the fetuses removed. Fetal lung sections were processed for light and electron microscopy. A group of animals treated with physiological saline in a similar manner served as the controls. The fetal lungs from treated animals were hypoplastic and retarded in development. The severity of pulmonary changes increased with the dose-levels of MMC. Vacuolation and lysis of mitochondria were seen in fetal lungs. Mitochondrial damage increased in severity with dose-level of methylmercuric chloride.     

Structural teratogenicity evaluation of methyl chloride in rats and mice after inhalation exposure

One hundred bred Fischer-344 female rats were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 25 females per exposure concentration, from gestation day (gd) 7 through gd 19. On gd 20, the females were sacrificed for evaluation of maternal reproductive and fetal parameters. Maternal and fetal toxicity was apparent at the highest exposure concentration. There were no methyl chloride-induced external, skeletal, or visceral abnormalities seen in the fetuses. One hundred thirty-two C57BL/6 female mice bred to C3H males to produce B6C3F1 offspring were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 33 females per exposure concentration, from gd 6 through gd 17. Exposure to the entire 1,500-ppm group was terminated on gd 10-14, with the animals killed in extremis. Selective necrosis of neurons in the internal granular layer of the cerebellum, ranging from individual cell involvement to focal areas comprising large numbers of neurons, was found in all females. On gd 18, the females from the other treatment groups, all of which survived, were killed for evaluation of maternal reproductive and fetal parameters. No evidence was seen of maternal or fetal toxicity in these exposure groups. There were no significant alterations in external appearance in fetuses from any of the exposure groups. Visceral examination of mouse fetuses revealed a small, but statistically significant, incidence of heart defects in litters of the 500-ppm group. The anomaly, a reduction or absence of the atrioventricular valve, chordae tendineae, and papillary muscle, was observed on the left side (bicuspid valve) in three fetuses and the right side (tricuspid valve) in six fetuses: three males and six females. It is concluded that methyl chloride inhalation exposure in pregnant rats, during critical periods of embryo and fetal development, is not teratogenic at concentrations which elicit maternal and fetal toxicity. In pregnant mice, methyl chloride was severely toxic to dams following 4 days or more of exposure to 1,500 ppm in air. Methyl chloride, at 500, but not 100 ppm, was teratogenic in mice, leading to a malformation in the heart. No embryo-fetal toxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 100 ppm of ethyl chloride.     

Effects of sodium valproate and oxygen on the CD-1 mouse fetus

This study reports the effects of valproic acid (VA) on the CD-1 mouse fetus when the drug is administered continuously via osmotic minipumps at human therapeutic drug plasma levels. Two VA-filled Alzet osmotic minipumps were implanted subcutaneously on gestation day 5 for continuous exposure of a total daily dosage of 850 mg/kg on gestation days 5-12. Dams were then exposed continuously to either normoxic (21% oxygen), hyperoxic (50% oxygen), or hypoxic (12% oxygen) controlled environments during gestation days 5-12, in order to determine if hyperoxic maternal conditions offered a protective environment for the fetus, and conversely, if hypoxia exacerbated teratogenicity. Dams were sacrificed on gestation day 18, and litter and fetal data were collected. It was determined in separate groups under normoxic conditions that the osmotic minipump system maintained VA plasma levels corresponding to human therapeutic levels. Sodium valproate was found to induce developmental toxicity in the CD-1 mouse fetus at human therapeutic drug plasma levels. Fetal weights were reduced, and the number of resorptions, deaths, and hematomas was increased. While hypoxia exacerbated the toxic effect on the fetus, hyperoxia failed to ameliorate the outcome.     

Developmental toxicity studies in Crl:CD (SD) rats following inhalation exposure to trichloroethylene and perchloroethylene

The potential for trichloroethylene (TCE) and perchloroethylene (PERC) to induce developmental toxicity was investigated in Crl:CD (SD) rats whole-body exposed to target concentrations of 0, 50, 150 or 600 ppm TCE or 0, 75, 250 or 600 ppm PERC for six hours/day, seven days/week on gestation day (GD) 6-20 and 6-19, respectively. Actual chamber concentrations were essentially identical to target with the exception of the low PERC exposure level, which was 65 ppm. The highest exposure levels exceeded the limit concentration (2 mg/L) specified in the applicable test guidelines. Maternal necropsies were performed the day following the last exposure. Dams exposed to 600 ppm TCE exhibited maternal toxicity, as evidenced by decreased body weight gain (22% less than control) during GD 6-9. There were no maternal effects at 50 or 150 ppm TCE and no indications of developmental toxicity (including heart defects or other terata) at any exposure level tested. Therefore, the TCE NOEC for maternal toxicity was 150 ppm, whereas the embryo/fetal NOEC was 600 ppm. Maternal responses to PERC were limited to slight, but statistically significant reductions in body weight gain and feed consumption during the first 3 days of exposure to 600 ppm, resulting in a maternal NOEC of 250 ppm. Developmental effects at 600 ppm consisted of reduced gravid uterus, placental and fetal body weights, and decreased ossification of thoracic vertebral centra. Developmental effects at 250 ppm were of minimal toxicological significance, being limited to minor decreases in fetal and placental weight. There were no developmental effects at 65 ppm.     

Circadian modification of ethanol damage in utero to mice

This study evaluates the ethanol toxicity for fetal development at different circadian stages. Pregnant mice were given a single intraperitoneal ethanol injection on day 7, 8, or 10 of gestation at one of four circadian stages (0700, 1300, 1900, or 0100 hr). The dams were killed on the day before term (day 18). Prenatal exposure to ethanol resulted in an increased number of resorptions, reduced fetal body weight, and produced an increased incidence of external alterations. The severity of damage was related to the dose, the period of gestation, and particularly to the circadian stage at the time of treatment. Ethanol had the greatest effect on the embryo of a mouse when administered at the mid-dark span. Consequently exposure to a single dose of ethanol at one time or another along the 24-hr time scale during organogenesis has important implications for the substantially increased risk.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.     

Gestational stage sensitivity to ultrasound effect on postnatal growth and development of mice

BACKGROUND: An experiment was conducted to find out whether ultrasound exposure leads to changes in postnatal growth and development in the mouse. METHODS: A total of 15 pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, 65 mW/cm2, I(SPTP) = 1 mW/cm2 Intensity(Spatial Peak-Temporal Peak), I(SATA) = 240 mW/cm2 Intensity(Spatial Average-Temporal Average)) for 30 min for a single day between days 10 and 18 of gestation (GD 10-18). Virgin female mice were placed with same age group males for mating in the ratio 2 females : 1 male and examined the next morning for the presence of vaginal plug, a sign of successful copulation. The females with vaginal plugs were separated and labeled as 0-day pregnant. Maternal vaginal temperature was also measured. A sham exposed control group of 15 pregnant mice was maintained for comparison. All exposed as well as control animals were left to complete gestation and parturition. Their offspring were used in our further studies. They were monitored during early postnatal life for standard developmental markers, postnatal mortality, body weight, body length, head length, and head width, and growth restriction was recorded up to 6 weeks of age. RESULTS: An exposure to ultrasound induced nonsignificant deviations in the maternal vaginal temperature or developmental markers. Significant low birth weight was observed in the present study, after exposure at GD 11, 12, 14, and 16. However, 14 and 16 days postcoitus during the fetal period appears to be the most sensitive to the ultrasound effect, in view of the number of different effects as well as severity of most of the observed effects when exposed on these gestation days. CONCLUSIONS: The results indicate that diagnostic ultrasound can induce harmful effects on mouse growth and development when given at certain critical periods of gestation.     

Effect of prenatal exposure to diagnostic ultrasound on the development of mice.

Pregnant Swiss albino mice were exposed to diagnostic ultrasound (3.5 MHz, approximately 65 mW) for 10 min on Day 3.5 (preimplantation period), 6.5 (early organogenesis period), or 11.5 (late organogenesis period) of gestation. Sham-exposed controls were maintained for comparison. Exposed as well as control fetuses were dissected out on the 18th day of gestation, and changes in total mortality, body weight, body length, head length, brain weight, sex ratio, and microphthalmia were recorded. Exposure on Day 3.5 of gestation resulted in a small increase in the resorption rate and a significant reduction in fetal body weight. A low fetal weight and an increase in the number of growth-retarded fetuses were produced by exposure on Day 6.5 postcoitus. A statistically nonsignificant increase in the incidence of microphthalmia was induced in fetuses exposed on Day 6.5 or Day 11.5 of gestation. These results indicate that ultrasound may have some adverse effects on the mouse embryos depending on the developmental stage at which the exposure occurred.     

Developmental Toxicity Studies with Atrazine and its Major Metabolites in Rats and Rabbits

Atrazine (ATR), hydroxyatrazine (OH‐ATR), and the three chloro metabolites of ATR (deethylatrazine [DEA], deisopropylatrazine [DIA], diaminochlorotriazine [DACT]) were evaluated for developmental effects in rats and rabbits. Three developmental toxicity studies were conducted on ATR in rats (two studies) and rabbits and a developmental toxicity study was conducted in rats for each of the four ATR metabolites DEA, DIA, DACT, and OH‐ATZ. ATR administration by gavage to pregnant rats and rabbits from implantation (gestation day [GD] 6 in rat, GD 7 in rabbit) through closure of the palate (GD 15 in rat and GD 19 in rabbit) did not statistically significantly alter the incidence of developmental abnormalities or malformations at dose levels up to 100 (rat) or 75 (rabbit) mg/kg bw/day. There were no effects on developmental toxicity parameters for DEA, DIA, DACT, or OH‐ATR at oral dose levels up to 100, 100, 150, or 125 mg/kg bw/day, respectively, with the exception of reductions in fetal body weight by DACT and OH‐ATR in the presence of decreased maternal body weight gain. ATR did not adversely affect developmental end points in a two‐generation study conducted in rats exposed to dose levels up to 500 ppm (38.7 mg/kg/day) in the diet. The 500‐ppm dose level resulted in significantly reduced maternal body weight gain. Overall, data show that neither ATR nor its metabolites statistically significantly affected rat or rabbit embryo‐fetal development even at dose levels producing maternal toxicity.     

Measurement of plasma or urinary metabolites and Hprt mutant frequencies following inhalation exposure of mice and rats to 3-butene-1,2-diol

Studies were performed to determine if the detoxification pathway of 1,3-butadiene (BD) through 3-butene-1,2-diol (BD-diol) is a major contributor to mutagenicity in BD-exposed mice and rats. First, female and male mice and rats (4-5 weeks old) were exposed by nose-only for 6h to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure concentrations that yielded similar plasma levels of BD-diol, and then animals were exposed in inhalation chambers for 4 weeks to BD-diol to determine the mutagenic potency estimates for the same exposure levels and to compare these estimates to those reported for BD-exposed female mice and rats where comparable blood levels of BD-diol were achieved. Measurements of plasma levels of BD-diol (via GC/MS methodology) showed that (i) BD-diol accumulated in a sub-linear fashion during single 6-h exposures to >200 ppm BD; (ii) BD-diol accumulated in a linear fashion during single or repeated exposures to 6-18 ppm BD and then in a sub-linear fashion with increasing levels of BD-diol exposure; and (iii) exposures of mice and rats to 18 ppm BD-diol were equivalent to those produced by 200 ppm BD exposures (with exposures to 36 ppm BD-diol yielding plasma levels approximately 25% of those produced by 625 ppm BD exposures). Measurements of Hprt mutant frequencies (via the T cell cloning assay) showed that repeated exposures to 18 and 36 ppm BD-diol were significantly mutagenic in mice and rats. The resulting data indicated that BD-diol derived metabolites (especially, 1,2-dihydroxy-3,4-epoxybutane) have a narrow range of mutagenic effects confined to high-level BD (>or=200 ppm) exposures, and are responsible for nearly all of the mutagenic response in the rat and for a substantial portion of the mutagenic response in the mouse following high-level BD exposures.     

Cadmium exposure on day 12 of gestation in the Wistar rat: distribution, uteroplacental blood flow, and fetal viability

The effects of cadmium exposure (40 mumole CdCl2/kg, s.c.) on day 12 of gestation were evaluated in the Wistar rat. At 16-18 hours following such cadmium exposure, blood flow (as determined by radiolabeled microspheres) to the chorioallantoic placenta (CAP) was significantly reduced by 35%; at 24-26 hours, blood flow to the CAP had returned to control levels and was still unaffected at 38-43 hours. Uterine blood flow was not significantly altered at any of these timepoints. Between 16-18 and 24-26 hours after cadmium exposure, the concentration of cadmium in the placenta decreased significantly, while total cadmium content did not change. By 38-43 hours after cadmium exposure, total cadmium content of the placenta had increased significantly, although cadmium concentration was unchanged. There were no adverse effects on fetal viability or growth, as determined on day 20 of gestation. In sharp contrast, near-term (day 18) exposure to 40 or 50 mumole CdCl2/kg (s.c.) resulted in 53% and 82% mean incidences of fetolethality, respectively, within 24 hours. Administration of 50 mumole CdCl2/kg (sc) on day 12 also had no effect on fetal growth but resulted in increased fetolethality (12%). Thus midgestational cadmium exposure and its accompanying alterations in placental blood flow do not compromise fetal viability or growth. The differential response to cadmium at mid- and late gestation, in terms of fetolethality, is not due to maternal cadmium dose.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat

Epidemiological evidence suggests that hypertension and coronary heart disease are programmed by exposure to a poor diet during intrauterine life. It has been proposed that the prenatal environment may exert an adverse effect on the development of the kidney and hence later control of blood pressure. These assertions are supported by animal experiments. In the rat, fetal exposure to a maternal low protein diet is associated with disproportionate patterns of fetal growth and later elevation of blood pressure. Pregnant female rats were fed control (18% casein) or low protein diets throughout pregnancy, or during specific periods. Nephron number was determined at day 20 gestation, full term and 4 weeks of age. Exposure to low protein throughout gestation, or in mid-late gestation increased total nephron number at day 20. By term nephron number was reduced, relative to controls, in rats that were undernourished between days 8-14 or 15-22 gestation. At 4 weeks postnatally rats exposed to low protein throughout fetal life had a reduced (13%) nephron complement and blood pressures 13 mmHg above control animals. Lower renal size and elevated blood pressure persisted to 19 weeks of age, at which time glomerular filtration rate was normal. The data are consistent with the hypothesis that maternal undernutrition may programme the renal nephron number and hence impact upon adult blood pressure and the development of renal disease.