The effect of low-level prenatal X-irradiation on postnatal development in the Wistar rat

The objective of this investigation was to determine the effect of low-dose prenatal X-irradiation on postnatal growth and neurobehavioral development, and whether alterations would manifest at dosages lower than those which produce anatomic malformations from exposure at the most sensitive period of organogenesis. Ninety-eight Wistar strain rats were exposed to 0.1, 0.2, or 0.4 Gy X-radiation of were sham irradiated on the 9th or 17th day of gestation. A conventional teratologic evaluation was completed on half of the animals (572 fetuses). The age of appearance of four physiologic markers and of acquisition of six reflexes was observed in 372 offspring. Exposure during early organogenesis at these levels had no effect on any of these parameters. Prenatal exposure to X-radiation on the 17th day of gestation at dosage levels greater than 0.1 Gy resulted in alterations in the appearance of three postnatal neurophysiologic parameters. Growth retardation throughout the postpartum period also was observed in the offspring. The induction of developmental and reflex alterations had a comparable threshold to the known threshold for anatomic malformations on the 9th day. These results indicate that all of the parameters studied had thresholds either at or above 0.2 Gy acute radiation, and that the postpartum developmental and reflex acquisition measures were not more sensitive indicators of exposure to X-radiation than growth parameters.     

Studies of the effect of 0.4-Gy and 0.6-Gy prenatal X-irradiation on postnatal adult behavior in the Wistar rat

Thirty-four pregnant Wistar rats were X-irradiated on the 9th or 17th day of gestation at a dosage level 0.4 Gy or 0.6 Gy or were sham-irradiated. All mothers were allowed to deliver their offspring, and litters were limited to a maximum of eight on day 2. On day 30, 224 offspring were weaned and raised until 60 days of age, at which time testing began. Each rat randomly received, in random order, three of the following six behavioral tests: Water T-maze, Conditioned Avoidance Response, Forelimb Hanging, Activity Wheel, Swimming, and Open Field. There were no statistically significant differences between the irradiated and control groups for maternal weight or weight gain or mean litter size, although the litter size of the 17th day 0.6-Gy group was slightly lower. Among offspring irradiated with 0.6 Gy on the 17th day, 3-day-old neonates' weights were significantly reduced. Offspring irradiated on the 17th day with 0.6 Gy exhibited higher Conditioned Avoidance Response 5th-day and retest avoidance scores than did the controls. There were also significant sex differences in responses within the irradiated and control groups for several tests, which were unrelated to radiation exposure. The results of this study indicate that low-level X-irradiation during the fetal period of rat gestation results in neonatal growth retardation and subtle behavioral alterations that may be manifested in adult life. Growth retardation may be the most sensitive indicator of subtle effects that result from low-level prenatal exposure to X-rays.     

Effects of prenatal X-irradiation on the 14th-18th days of gestation on postnatal growth and development in the rat

Thirty-nine pregnant adult Wistar strain rats were randomly assigned to one of three exposure groups: 0, 0.75, or 1.50 Gy X-radiation total exposure. Animals were exposed from the 14th to the 18th days of gestation at 0, 0.15, or 0.30 Gy per day. At term, 15 rats were killed and morphologic analyses were completed. Twenty-four rats were allowed to deliver their offspring. On the first day of postnatal life, litters were reduced to a maximum of eight pups per litter, with equal numbers of male and female offspring wherever possible. A total of 187 pups were observed for the age of acquisition of five reflexes (air righting, surface righting, visual placing, negative geotaxis, auditory startle) and the appearance of four physiologic markers (pinna detachment, eye opening, vaginal opening, testes descent). There was significant dose-related weight reduction in term fetuses and offspring throughout the 86-day postnatal period. Postnatal growth rate (g gained/day) was unaffected. Adult offspring brain and gonadal weight and organ weight:body weight ratios were reduced. Using the PAC50 methodology, dose-related alterations occurred in the acquisition of several reflexes. All physiologic markers exhibited a dose-related delay in appearance. These results indicate that fractionated exposure to X-radiation during the fetal period in the rat results in dose-dependent alterations in postnatal growth and physiologic development. These studies are important for our understanding of the long-range effects of prenatal exposure to ionizing radiation late in gestation.     

Effects of 0.6-Gy prenatal X irradiation on postnatal neurophysiologic development in the Wistar rat

Forty-one pregnant Wistar strain rats were irradiated with 0.6-Gy X rays or were sham irradiated on the 9th or 17th days of gestation to determine if this dosage level would result in alterations in postnatal neurophysiologic development. Half of the mothers were sacrificed at term, and the developmental status of 221 newborns was evaluated. The remaining mothers delivered and raised their litters. The 161 offspring were observed for the age of attainment of the following physiologic parameters: pinna detachment, eye opening, testes opening. Offspring were also tested for the acquisition of the following selected reflexes: surface righting, negative geotaxis, auditory startle, air righting, and visual placing. Term fetal weight was lower than the controls in the group irradiated on the 9th day but was recuperable postnatally. None of the 9 developmental tests performed postnatally were abnormal in the animals irradiated on the 9th day. Thus, at least with regard to these measures, the surviving embryos exposed during the all-or-none period could not be differentiated from the controls. Offspring irradiated on the 17th day exhibited retarded growth which persisted during neonatal life. The three-day-mean neonatal weight was significantly lower in the group irradiated on the 17th day compared to controls. There were no significant maternal body weight or organ/weight differences between the groups. Rats exposed in utero on the 17th day had a significantly delayed acquisition of air righting. These results demonstrate that 0.6-Gy in utero irradiation on the 17th day of gestation can cause subtle alterations in growth and development of the Wistar strain rat during postnatal life.     

Effects of prenatal X-irradiation on postnatal testicular development and function in the Wistar rat: development/teratology/behavior/radiation

It is evident that significant permanent tissue hypoplasia can be produced following radiation exposure late in fetal development. Because two organs, brain and testes, are developmentally and functionally interrelated, it was of interest to determine whether fetal testicular hypoplasia was a primary or a secondary effect of fetal brain irradiation. Twenty-four pregnant Wistar strain rats were randomly assigned to one of four groups, and a laparotomy was performed on day 18 of gestation. The fetuses received sham irradiation, whole body irradiation, or only head/thorax or pelvic body irradiation at a dosage level of 1.5 Gy. Mothers were allowed to deliver and raise their offspring until postnatal day 30, when the offspring were weaned. At 60 days of age, 74 male offspring were allowed to mate with colony control females of similar age until successful insemination or until the males reached 90 days of age, when they were killed. Testes were weighed and processed for histologic examination. Direct radiation of testes, due to whole body or pelvic exposure, resulted in testicular growth retardation and significantly reduced spermatogenesis. Breeding activity of the males and the percent of positive inseminations were also slightly reduced. However, a significant percentage of male offspring receiving direct testicular radiation did produce offspring. Head/thorax-only irradiation did not adversely affect testicular growth or spermatogenesis. Therefore, the use of histologic analysis as the sole determinant of infertility may be misleading. This study indicates that testicular growth retardation and an increased infertility rate result from direct prenatal exposure of rat testes to X-radiation and are not necessarily mediated via X-irradiation effects on the central nervous system.     

The effects of prenatal X irradiation on the appearance of reflexes and physiologic markers in the neonatal rat

Seventy pregnant adult Wistar strain rats were randomly assigned to 1 of 12 exposure groups; 9th or 17th day irradiation at the 0-, 0.1-, 0.2-, 0.4-, 0.6-, or 0.8-Gy dosage level. On the first day of postnatal life, litters were reduced to a maximum of eight pups per litter. A total of 508 pups were observed for the age of acquisition of five reflexes (air righting, surface righting, visual placing, negative geotaxis, auditory startle) and the appearance of four physiologic markers (pinna detachment, eye opening, vaginal opening, testes descent). A dose-response relationship for alterations in reflex acquisition and physiologic marker appearance was observed due to exposure above 0.2 Gy on the 17th day of gestation. Therefore, 0.2 to 0.4-Gy exposure may represent a threshold range for exposure on the 17th day using these postnatal parameters.     

Maternal diazepam exposure on brain ornithine decarboxylase and growth of offspring

The prenatal treatment of diazepam on the developmental pattern of brain ornithine decarboxylase and the general growth of offspring were studied. Diazepam (120 mg/kg/day) was administered orally to pregnant Sprague-Dawley rats from day 14 to day 20 of gestation. The activity of brain ornithine decarboxylase and body weight of the offspring were measured from the late fetal stage to the early postnatal stage. It was found that diazepam inhibited both the prenatal and 4-hour postnatal ornithine decarboxylase activities, though the general maturation pattern of the enzyme in the brain was not much altered. It may indicate that diazepam inhibits early brain development. The enzyme activity fell off as it reached maturation. Prenatal treated neonates of 6-hour or older age group had the normal activities of brain ornithine decarboxylase. The general growth of the treated offspring was substantially retarded. Their body weights were very much lower than the control offspring. The results of the present study is an additional evidence that diazepam and other benzodiazepines should be used with great care in pregnant women.     

Adaptive response in embryogenesis: II. Retardation of postnatal development of prenatally irradiated mice.

We previously reported that a priming dose of 0.3 Gy on gestation day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations caused by exposure to 5 Gy X rays on gestation day 12 in ICR mice. In the present study, postnatal development of the live offspring was investigated using a set of developmental and behavioral parameters. The offspring of the mice irradiated with 0.3 Gy generally showed a delay in the appearance of most of the physiological markers, impaired acquisition of neonatal reflexes, and alteration of adult behavior. However, an increase in body weight in the females was observed 4 weeks postnatally. In the offspring primed with 0.3 Gy followed by a challenging dose of 5 Gy prenatally, a high postnatal mortality was found, and all the survivors had various radiation-induced detrimental effects. The results indicated that the priming dose was advantageous to survival itself, but was disadvantageous to the health of survivor. The results also suggested that studying the whole animal can show the extent of the effects of radiation, i.e. quality of life, in a way that cellular or molecular studies cannot.     

Reduced weight in mice offspring after in utero exposure to 2450-MHz (CW) microwaves

Time-bred CD-1 mice (100) were sham-irradiated or irradiated with 2450-MHz (CW) microwaves at 28 mW/cm² for 100 minutes daily from the 6th through 17th day of gestation. The offspring were examined either as fetuses after hysterotomy on the 18th day of gestation or as naturally born neonates on the 1st and 7th day of age. Fetuses of half of the dams were examined on the 18th day of gestation. The incidence of pregnancy and the numbers of live, dead, resorbed, and total fetuses were similar in both groups. The mean weight was significantly lower (10%) in live microwave-irradiated fetuses, and ossification of sternal centers was significantly delayed. In the offspring that were born naturally, the mean weight of microwave-irradiated 7-day-old suckling mice was significantly lower (10%) than that of the sham-irradiated group. Survival rates of neonates in these two groups were not different. These data demonstrate that the decreased fetal weight seen in microwave-irradiated mice is retained at least 7 days after birth. Evidence from other published studies is presented to show that the retarded growth is persistent and might be interpreted as permanent stunting.     

Effects of carbon-ion radiation on the postnatal development of mice and on the yield of white spots in the mid-ventrum and tail tips

Pregnant female C57BL/10JHir mice were irradiated whole-body at 9 days of gestation with a single acute dose of carbon-ion radiation. The average linear energy transfer (LET) of the carbon ions was 50 keV/microm within a spread-out Bragg peak (SOBP). The effects were studied by scoring changes in the postnatal development of the mice as well as in the pigmentation of the cutaneous coats and tail tips of their offspring 22 days after birth. The percentage of live births was reduced in mice exposed to carbon ions at doses greater than 0.5 Gy. The survival to day 22 was also reduced in mice exposed to carbon ions at doses greater than 0.75 Gy. Moreover, the body weight at day 22 was reduced in mice exposed to carbon ions at doses greater than 0.1 Gy. A comparison of the survival to day 22 after exposure to carbon ions with our previous results for 60Co gamma rays indicated that carbon ions were twice as effective as gamma rays. White spots were found in the mid-ventrum as well as in the tail tips of offspring exposed to carbon ions in utero. The frequency and the size of the white spots in the mid-ventrum and in the tail tips increased as the dose increased. Carbon ions appear to be slightly more effective than the gamma rays used in our previous study. In the ventral white spots, no melanocytes were observed in the epidermis, dermis and hair follicles. These results indicate that prenatal exposure to carbon ions has a greater effect on the postnatal development and survival of mice than does exposure to gamma rays, and that the relative biological effectiveness is greater than that for effects on melanocyte development.     

Effects of Podophyllum hexandrum on radiation induced delay of postnatal appearance of reflexes and physiological markers in rats irradiated in utero.

Effect of 2.0 Gy gamma-dose delivered to rats in utero on 17th day of gestation was studied to monitor the radiation induced retardation of neurophysiological development in postnatal young ones. Rhizome of Podophyllum hexandrum which has been well documented for mitigating radiation injuries in adult mice was attempted for modifying radiation damage. Rats were observed from postnatal day 1 to 25 for the age of the appearance of physiological markers (pinna detachment, inscisor's eruption, eye opening) and acquisition of reflexes (surface righting, visual placing, reflex suspension, negative geotaxis). In irradiated groups there was a significant weight reduction in mother rats and offsprings throughout the experimental period. There was radiation-induced delay in the appearance of pinna detachment but not in eye opening and inscisor's eruption. Appearance of the reflexes were also delayed due to irradiation. Preirradiation administration of the extract of Podophyllum hexandrum (i.p., 200 mg/kg/b.w.) mitigated radiation induced postnatal physiological alterations. These studies have implications in protection against damage (in utero) due to planned radiation exposure.     

Effect of prenatal alprazolam exposure on anxiety patterns in rat offspring

Prenatal alprazolam (APZ) treatment in 0.1 and 0.2 mg/kg/day doses during 13-20 days of gestation induced significant increase in open-field ambulation, rearings, self-grooming and faecal pellets in rat offspring. Prenatal APZ treated rats displayed significantly increased anxiogenic behaviour on elevated plus maze (spent less time on open arms, more time on enclosed arms and made less number of entries on open arms) and increased anxiogenecity on elevated zero maz e(APZ treated rats spent less time on open arms and made less number of head dips and stretched attend postures in comparison to control rat offspring). The results indicate persistent behavioural alterations in the rat offspring after prenatal exposure to APZ.     

Peculiarities of the ovary structure in the rat offspring developing under conditions of the removed thyroid gland of female rat with the thyroxine replacement therapy and external radiation exposure in utero

The influence of a single external gamma-radiation at a dose of 1.0 Gy (dose rate 9.08 x 10(-4) Gy/sec) on the 15th day of gestation in case of the removed complex of thyroid and parathyroid glands (thyroidparathyroidectomy) on the first day of gestation, as well as introduction of thyroxin and CaC12 on the structure of offspring ovary in postnatal ontogenesis (30-day old animals) was studied. It has been shown that thyroidparathyroidectomy of a female mother rat with thyroxine replacement therapy and irradiation, as well as the combination of these factors disturb the structure of ovarian tissues of the offspring. A single external irradiation on the 15th day of embryogenesis causes death of a considerable part of primordial follicles in the offspring ovary and growth of follicular layers in the secondary follicles. Thyroidparathyroidectomy of female rat on the first day of gestation with thyroxine replacement therapy causes delay in the development of follicles in the ovary at the early stages of maturation of 30-day old animals. The radiosensitivity of the ovarian tissues of the offspring that has been developed under the combined effect of the factors studied increases and results in an almost full loss of pool cells in the ovary of infant rats.     

Development of a useful technique for analyzing behavioral teratologic data

Two measures of postnatal development are described in this paper: the PAC50 and AD50. These measures proved to be more sensitive than the use of means in the evaluation of three radiation studies involving postnatal developmental evaluation. PAC50 is the percent of achievement of a goal by litters or offspring in an experimental group at the age when 50% of the control litters or offspring attain that goal. AD50 is the age (acquisition day) at which 50% of the litters or offspring in each group attain a specified developmental goal. This methodology is a useful technique for analyzing selected behavioral data following in utero X-irradiation and may prove to be a sensitive means of determining postnatal alteration due to prenatal exposure to a variety of suspect agents.     

The effect of ethanol exposure in pregnancy on maturation of monoaminergic systems in the developing rat bran

Simultaneous study of the main neurotransmitter of monoaminergic system of the brain, its metabolites, activity of catechol-O-methyltransferase (COMT) and the state of different subtypes of dopamine (DA) receptors in the developing brain of offspring from mothers alcoholized in gestation and feeding periods revealed a decrease in activity of all monoaminergic systems studied with reduction of noradrenaline and DA level in alcoholized fetus as well as of mPNA of COMT, an enzyme of catecholamine metabolism, in the structures of the forebrain on the 17th day but not on 13th day of prenatal development. In parallel experiments, an increase of the contents of both long and short splice variants of D2 DA receptor was registered. In postnatal period (days 4, 10, 17), further decrease of the DA system activity was observed, particularly a reduction of DOPAC level and DOPAC/DA ratio in rat litter, mothers of whom took alcohol in the gestation period with withdrawal it after birth of offspring. The serotonin system activity was also reduced in alcoholized litter in the postnatal period and was registered in the early stages (on the 4th day of life). Therefore, the serotonin system activity is changing at early stages of development (the 4th day), whereas inhibition of the DA system activity is registered at later stages (the 10th day of life).     

Prenatal dexamethasone administration disrupts the pattern of cellular development in rat lung

To examine whether prenatal exposure to glucocorticoids could adversely affect subsequent cellular development of the lung, we administered 0.2 mg/kg of dexamethasone to pregnant rats on gestational days 17, 18, and 19. Lungs of the offspring were then examined for patterns of cell acquisition (DNA) and growth (protein). DNA concentration (a marker of cell packing density) and DNA content (a measure of total cell numbers) were reduced during gestation, and the shortfalls in concentration persisted past weaning. Disruption of development was also apparent in the protein/DNA ratio, which was consistently elevated, a finding consistent with cellular hypertrophy. In addition, lung ODC became coupled to beta-adrenergic receptors prematurely in the dexamethasone group, suggesting that neural control of tissue differentiation is altered. These data indicate that prenatal glucocorticoids may compromise lung development through effects on cell replication and differentiation, which derive, in part, from alterations in the reception of trophic neural signals.     

Thyroid hormone levels in rats exposed to alcohol during development

Maternal ingestion of alcohol appears to cause a pattern of congenital anomalies with a reduction of pre- and postnatal growth in the offspring. In order to study the possible implication of thyroid function in the effects of pre- and/or postnatal exposure to alcohol, we have studied serum thyroxine (T4) and triiodothyronine (T3) levels in rats from alcohol-fed mothers during the postnatal period (0-50 days). Blood alcohol levels of ethanol-treated pregnant rats were approximately equal to 20-25 mM and their serum T4 levels were decreased, compared with the pair-fed controls, at 15 and 21 days of gestation. No significant changes were observed in T3 levels. Prenatal alcohol exposure was associated with a decrease in both T4 and T3 levels in pups at birth. Although T4 levels continued reduced in the 40-50 days of the postnatal period, no clear effects were observed on T3 levels during this time. Moreover, the more marked alterations were obtained when the offspring were postnatally and pre + postnatally exposed to alcohol. Significant decreases were found in both T4 and T3 levels following postnatal exposure, except at the 20-25th day when a marked but transient increase in T4 levels was observed. These results indicate that alcohol exposure disturbs the hypothalamo-pituitary-thyroid axis, as measured by T3 and T4 hormone levels, mainly when the rats are exposed during the postnatal period.     

Sedentary behavior during postnatal life is determined by the prenatal environment and exacerbated by postnatal hypercaloric nutrition

The discovery of a link between in utero experience and later metabolic and cardiovascular disease is one of the most important advances in epidemiology research of recent years. There is now increasing evidence that alterations in the fetal environment have long-term consequences on metabolic and endocrine pathophysiology in adult life. This process has been termed "fetal programming," and we have shown that undernutrition of the mother during gestation leads to obesity, hypertension, hyperphagia, hyperinsulinemia, and hyperleptinemia in offspring. Using this model of maternal undernutrition throughout pregnancy, we investigated whether prenatal influences may lead to alterations in postnatal locomotor behavior, independent of postnatal nutrition. Virgin Wistar rats were time mated and randomly assigned to receive food either ad libitum (ad libitum group) or at 30% of ad libitum intake (undernourished group). Offspring from UN mothers were significantly smaller at birth than AD offspring. At weaning, offspring were assigned to one of two diets [control or hypercaloric (30% fat)]. At ages of 35 days, 145 days, and 420 days, voluntary locomotor activity was assessed. At all ages studied, offspring from undernourished mothers were significantly less active than offspring born of normal birth weight for all parameters measured, independent of postnatal nutrition. Sedentary behavior in programmed offspring was exacerbated by postnatal hypercaloric nutrition. This work is the first to clearly separate prenatal from postnatal effects and shows that lifestyle choices themselves may have a prenatal origin. We have shown that predispositions to obesity, altered eating behavior, and sedentary activity are linked and occur independently of postnatal hypercaloric nutrition. Moreover, the prenatal influence may be permanent as offspring of undernourished mothers were still significantly less active compared with normal offspring at an advanced adult age, even in the presence of a healthy diet throughout postnatal life.     

Evaluation of the developmental effects on mice after prenatal, or pre- and postnatal exposure to 2,3-dimercaptopropane-1-sulfonic acid (DMPS)

The sodium salt of 2,3-dimercaptopropane-1-sulfonic acid (DMPS), a water soluble metal complexing agent, was administered to four groups of pregnant Swiss mice at 0, 70, 210, and 630 mg/kg/day by two dosing schedules: gestation day 14 until birth (prenatal exposure), and gestation day 14 until postnatal day 21 (pre- and postnatal section). Dams were allowed to deliver and the number of live and dead pups recorded. Each pup was sexed and weighed on days 0, 4, 14, and 21. Also, pinna detachment, incisor eruption and eye opening were monitored. No adverse effects on offspring survival or development were evident in either exposures at doses employed in this study. The "no observable effect level" (NOEL) for health hazard to the developing fetus or pup was 630 mg DMPS/kg/day. This dose is much higher than the amounts of DMPS usually administered in human heavy metal poisoning.     

Effects of prenatal exposure to low doses of diethylstilbestrol, o,p'DDT, and methoxychlor on postnatal growth and neurobehavioral development in male and female mice

We examined effects of a wide range of doses of three man-made estrogenic chemicals during fetal life on neurobehavioral changes during early postnatal life in mice. Pregnant mice were fed a 4-log range of o,p'DDT, methoxychlor (MXC), and the drug diethystilbestrol (DES) from gestation days 11 to 17. Offspring were examined for changes in postnatal growth and the development of neuromuscular reflexes. Fetal exposure to the estrogenic chemicals altered the number of live pups per litter, the sex ratio of the litters, the anogenital distance of male and female offspring at birth (a bioassay for fetal androgen action), and the body weight of offspring at birth and during the first 5 days of postnatal life. In most cases, however, the dose-response relationships were complex (non-monotonic), with effects at the highest dose examined being opposite to effects seen at lower doses. The two markers of neurobehavioral development, righting and cliff avoidance reflexes, were not sensitive indicators of prenatal estrogen exposure. Only maternal exposure to the lowest MXC dose produced an increase in reactivity in righting and cliff avoidance tests in offspring.