Exposición a contaminantes atmosféricos durante el embarazo y desarrollo prenatal y neonatal: protocolo de investigación en el proyecto INMA (Infancia y Medio Ambiente) / Air pollutant exposure during pregnancy and fetal and early childhood development. Research protocol of the INMA [Childhood and Environment Project]

Introducción: El proyecto INMA (Infancia y Medio Ambiente) es una red de investigación cooperativa que tiene como objetivos estudiar los efectos del medio ambiente y la dieta en el desarrollo fetal e infantil. El objetivo de este artículo es presentar el protocolo de exposición a contaminantes atmosféricos durante el embarazo y desarrollo prenatal y neonatal en el proyecto INMA. Métodos: La información para la evaluación de la exposición a contaminación atmosférica durante el embarazo se basa en mediciones de contaminantes atmosféricos en el exterior (dióxido de nitrógeno [NO2], compuestos orgánicos volátiles [COV], ozono, partículas [PM10, PM2,5] y su composición [hidrocarburos aromáticos policíclicos]), medición de contaminantes de exposición individual (en el interior de la vivienda y captadores personales [COV y NO2]), determinación de un marcador biológico de exposición a hidrocarburos (1-hidroxipireno), en información recogida mediante cuestionarios y en la utilización de sistemas de información geográfica. Esta información permite elaborar índices de exposición individual a contaminación atmosférica con los que analizar su posible relación con el desarrollo fetal y la salud del recién nacido. Discusión: El protocolo que se presenta y el tipo de estudio permiten obtener una aproximación a la exposición individual a contaminantes atmosféricos. Por último, el elevado número de participantes (n = 4.000), así como la heterogeneidad de las características ambientales y sociodemográficas, acrecienta el potencial del estudio

Introduction: The INMA (INfancia y Medio Ambiente [Spanish for Environment and Childhood]) project is a cooperative research network. This project aims to study the effects of environment and diet on fetal and early childhood development. This article aims to present the air pollutant exposure protocol during pregnancy and fetal and early childhood development of the INMA project. Methods: The information to assess air pollutant exposure during pregnancy is based on outdoor measurement of air pollutants (nitrogen dioxide [NO2], volatile organic compounds [VOC], ozone, particulate matter [PM10, PM2,5 ] and of their composition [polycyclic aromatic hydrocarbons]); measurement of indoor and personal exposure (VOC and NO2); urinary measurement of a biological marker of hydrocarbon exposure (1-hydroxypyrene); and data gathered by questionnaires and geographic information systems. These data allow individual air pollutant exposure indexes to be developed, which can then be used to analyze the possible effects of exposure on fetal development and child health. Conclusion: This protocol and the type of study allow an approximation to individual air pollutant exposure to be obtained. Finally, the large number of participants (N = 4,000), as well as their geographic and social diversity, increases the study's potential

Neurobehavioral and cognitive performances of children exposed to low-dose radiation in the Chernobyl accident: the Israeli Chernobyl Health Effects Study.

Exposure to low levels of ionizing radiation after the Chernobyl accident in the Ukraine could potentially have influenced the neurobehavioral and cognitive performances of exposed children. A cohort study of adolescents who were children at the time of the accident and who subsequently emigrated to Israel was conducted in 1998-2001. A total of 1,629 children (59% of all 2,769 invited) were included in the study (41% from higher contamination areas, 25% from lower contamination areas, 34% from noncontaminated areas). Mean scores of the Raven Standard Progressive Matrices Test were highest in children in all exposure groups whose parents had a high level of education. No overall relation was found between the cognitive function scores of the child and his/her putative radiation exposure level. Conners' test T scores did not differ significantly by level of exposure. Mothers of all exposure groups who were pregnant at the time of the accident gave their children significantly higher Conners' test scores than did those who were not pregnant. Scores for hyperactivity and attention-deficit/hyperactivity disorder were significantly higher among those who were in utero at the time of the accident. These results do not show differences of neurobehavioral or cognitive performance in exposed versus nonexposed children. There is a possible behavioral effect among offspring of pregnant mothers or mothers of very young children in all exposure levels.

Absence of harmful effects of magnetic resonance exposure at 1.5 T in utero during the third trimester of pregnancy: a follow-up study.

In this study the possible adverse effects of in utero exposure to magnetic resonance (MR) conditions at 1.5 Tesla were examined. Thirty-five children between 1 and 3 years of age, and nine children between 8 and 9 years of age, that were exposed to MR during the third trimester of pregnancy, were checked for possible adverse effects in a follow-up study. Data on pregnancy and birth, the results of a neurological examination at 3 months, their medical documentary with emphasis on eye and ear functioning, and a questionnaire answered by their mothers were collected and evaluated. In five children abnormal test results were observed, that had no relation to the MR exposure. No harmful effects of prenatal MR exposure in the third trimester of pregnancy were detected in this study.

[Modifying influence of iodine deficiency on radiation damage to the fetus (review)]. / Modifitsiruiushchee vliianie iodnogo defitsita na radiatsionnye porazheniia ploda (obzor).

It known an oppressing action of radiation, including radioactive iodine isotopes on the reproductive system and fetus development. There are clinical data on a negative influence of iodine deficiency on the course of pregnancy and fetus development resulting from hormonal disfunction of thyroid gland and a mother-fetus system. There are no data about a character and mechanisms of interaction of radiation and iodine endemia at the combined action on the gonads and fetus. The urgency of this problem is caused by the fact that many regions of the country are characterized to some extent by iodine deficiency in local food and water (in Russian Federation such regions make approximately 50% of territories), and the opportunity of radiation accidents at nuclear plants with contamination of the environment with products of nuclear division (significant part of which is radioactive iodine isotopes is an objective reality. The analysis of a few published and own experimental data allows us to conclude that the combined influence of an external gamma-irradiation and iodine deficiency on reproductive function has a synergic character.

Atm-null mice exhibit enhanced radiation-induced birth defects and a hybrid form of embryonic programmed cell death indicating a teratological suppressor function for ATM.

ATM (ataxia-telangiectasia mutated) is a genotoxic stress transducer. In this first report of Atm-dependent birth defects, Atm-null embryos were uniquely susceptible to low-dose (0.5 Gy) radiation, exhibiting severe runting, tail anomalies, and lethality, independent of cell cycle arrest or insulin-like growth factor 1. This treatment enhanced levels of p53 protein and central nervous system (CNS) apoptosis in wild-type mice, but not Atm-null mutants, at 6 h postirradiation. At 48 h, however, this pattern was reversed, with Atm-null mice exhibiting high levels of a hybrid form of programmed cell death within the CNS. Even heterozygous Atm-deficient embryos were radiosensitive to a higher radiation dose of 2 Gy. These results show that Atm is a novel teratologic suppressor gene protecting embryos from pathological cell death and teratogenesis initiated by even mild DNA damage.

Adaptive response in embryogenesis: V. Existence of two efficient dose-rate ranges for 0.3 Gy of priming irradiation to adapt mouse fetuses.

The adaptive response is an important phenomenon in radiobiology. A study of the conditions essential for the induction of an adaptive response is of critical importance to understanding the novel biological defense mechanisms against the hazardous effects of radiation. In our previous studies, the specific dose and timing of radiation for induction of an adaptive response were studied in ICR mouse fetuses. We found that exposure of the fetuses on embryonic day 11 to a priming dose of 0.3 Gy significantly suppressed prenatal death and malformation induced by a challenging dose of radiation on embryonic day 12. Since a significant dose-rate effect has been observed in a variety of radiobiological phenomena, the effect of dose rate on the effectiveness of induction of an adaptive response by a priming dose of 0.3 Gy administered to fetuses on embryonic day 11 was investigated over the range from 0.06 to 5.0 Gy/min. The occurrence of apoptosis in limb buds, incidences of prenatal death and digital defects, and postnatal mortality induced by a challenging dose of 3.5 Gy given at 1.8 Gy/min to the fetuses on embryonic day 12 were the biological end points examined. Unexpectedly, effective induction of an adaptive response was observed within two dose-rate ranges for the same dose of priming radiation, from 0.18 to 0.98 Gy/ min and from 3.5 to 4.6 Gy/min, for reduction of the detrimental effect induced by a challenging dose of 3.5 Gy. In contrast, when the priming irradiation was delivered at a dose rate outside these two ranges, no protective effect was observed, and at some dose rates elevation of detrimental effects was observed. In general, neither a normal nor a reverse dose- rate effect was found in the dose-rate range tested. These results clearly indicated that the dose rate at which the priming irradiation was delivered played a crucial role in the induction of an adaptive response. This paper provides the first evidence for the existence of two dose-rate ranges for the same dose of priming radiation to successfully induce an adaptive response in mouse fetuses.

Endogenous electric fields in embryos during development, regeneration and wound healing.

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm(-1) was measured near the posterior intestinal portal in 2-4 day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm(-1) across the primitive streak. Mammalian skin wounds generate 150 mV mm(-1) fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm(-1). The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m(-1).

Rapporteur report: other tissues.

This report covers the session devoted to 'other tissues'. It considers the effects of internal electric fields such as those induced by exposure to weak, extremely low frequency (ELF) electromagnetic fields, on cardiac physiology, neuroendocrine (pineal) function and on the processes of tissue repair and embryonic development. Summaries are provided for each of the papers presented, and the major aspects of the plenary session are discussed. Overall, these tissues and processes were not considered to be sensitive to the direct effects of weak ELF fields, although indirect effects may occur via field induced changes to the central nervous system.

Radiofrequency fields and teratogenesis.

Experimental studies that sought teratologic effects or developmental abnormalities from exposure to radiofrequency electromagnetic fields (RFEMF) in the range 3 kHz-300 GHz are critically reviewed for their possible consequences on human health. Those studies were conducted on beetles, birds, rodents, and nonhuman primates. Collectively, those experimental studies indicate that teratologic effects can occur only from exposure levels that cause biologically detrimental increases in body temperature. No reliable experimental evidence was found for nonthermal teratologic effects; rodents, mouse fetuses, and perinatal mice are more susceptible to such effects than rats. The primary confirmed effect in rats at high RFEMF levels was initial weight deficits in fetuses and neonates that decreased with infant growth. More generally from findings with pregnant mammals, exposures at RFEMF levels far higher than those permitted under the IEEE human exposure guidelines are necessary to reach or exceed cited experimental thresholds for maternal temperature increases. Some results indicated that the levels necessary to cause such effects in pregnant mammals could exceed those lethal to the dams. In a behavioral study of squirrel monkeys, no effects were observed on usual dam-offspring interactions or EEGs, but unexpected deaths of a number of offspring had occurred. However, this finding was not confirmed in a study solely on infant death using a larger number of subjects for greater statistical validity. Also reviewed were epidemiologic studies of various human populations considered to have been chronically exposed to environmental levels of RFEMF. Early studies on the incidence of congenital anomalies yielded no credible evidence that chronic exposure of pregnant women or of fathers exposed to RFEMF from nearby sources at levels below those guidelines would cause any anomalies in their offspring. The findings of studies on pregnancy outcomes of female physiotherapists occupationally exposed while treating patients with RFEMF were mixed, but taken collectively, the findings were negative.

[Reaction of posterity of two generations after exposure of pregnant Wistar rats to low dose irradiation during the period of fetus formation. Development of the first generation posterity]. / Posledstviia dlia potomstva dvukh pokolenii oblucheniia beremennykh samok-krys Vistar v malykh dozakh v period zakladki reproduktivnoi sistemy plodov. Razvitie potomstva pervogo pokoleniia.

Whole-body single exposure of female Wistar rats to 0.25, 0.5 and 1 Gy of gamma-rays (dose rate of 0.03 cGy/s) on the 10th day of pregnancy (a period of formation of the reproductive system in fetus) was carried out. To study irradiation consequences on the antenatal and postnatal development of the progeny 220 females, 700 19-day-old fetuses and about 1100 young rats were examined. The antenatal development of the progeny of the first generation was significantly impaired after the exposure to 1 Gy. However even less radiation doses resulted in a pronounced tendency to higher rates of intrauterine death and a lower number of live fetuses. Significant deviatins in the postnatal development of the first generation progeny were found after the exposure to 0.5 Gy, although the exposure to 0.25 Gy led to a higher rate of postnatal death and a less number of newborns in the litter.

Atm and c-Abl cooperate in the response to genotoxic stress during nervous system development.

The c-Abl proto-oncogene is a target of the ATM kinase after DNA double strand breaks, although the physiological significance of these signaling events is not clear. Therefore, to delineate the roles of c-Abl and Atm during mouse development we generated mice with combinations of c-Abl and Atm mutant alleles. We found that dual inactivation of Atm and c-Abl usually resulted in midgestational lethality. However, mice with three mutant alleles, c-Abl(-/-)Atm(+/-) or c-Abl(+/-)Atm(-/-), were viable but predisposed to neuro-developmental abnormalities after genotoxic insult. Thus, these genetic data link Atm and c-Abl signaling and underscore a significant interrelationship between the two during neural development.

Developmental toxicity evaluation of ELF magnetic fields in Sprague-Dawley rats.

To identify possible effects of horizontally polarized magnetic field (MF) exposure on maintenance of pregnancy and embryo-fetal development, an MF exposure system was designed and constructed and 96 time-mated female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz MF at field strengths of 0 (sham control) and 5, 83.3, or 500 microT (50, 833, or 5000 mG). Dams received MF or sham exposures for 22 h/day on gestational day 6-20. MF was monitored continuously throughout the study. There were no evidences of maternal toxicity or developmental toxicity in any MF exposed groups. Mean maternal body weight, organ weights, and hematological and serum biochemical parameters in groups exposed to MF did not differ from those in sham control. No exposure related differences in fetal deaths, fetal body weight, and placental weight were observed between MF exposed groups and sham control. External, visceral, and skeletal examination of fetuses demonstrated no significant differences in the incidence of fetal malformations between MF exposed and sham control groups. In conclusion, exposure of pregnant rats to 60 Hz at MF strengths up to 500 microT during gestation day 6-20 did not produce any biologically significant effect in either dams or fetuses.

Fetal dose during radiotherapy: clinical implementation and review of the literature.

Two pregnant patients received radiation therapy, one for the treatment of mediastinal Hodgkin's lymphoma and the other for a head and neck squamous cell carcinoma. The fetuses were both protected by additional shielding which reduced the unshielded exposure of the first fetus by 20-40%, and that of the second by 20-60%. The first child received an estimated maximum dose of 42 cGy, the second a maximum dose of 9 cGy. Treatment details are reported and a review of the literature that addresses the possible irradiation-induced side effects at low doses is included.

[Embryogenesis and early postnatal ontogenesis of posterity of two generations of female Wistar rats, depending on the time of their fertilization after low dose radiation exposure]. / Embriogenez i rannii postnatal'nyi ontogenez potomstva dvukh pokolenii samok krys Vistar v zavisimosti ot vremeni ikh oplodotvoreniia posle oblucheniia v malykh dozakh.

On the first day and 3-10 or 40-60 days after a single whole-body gamma-irradiation with doses of 0.25, 0.5 and 1 Gy, the pubertal female Wistar rats coupled with intact males. The embryogenesis and early postnatal ontogenesis of posterity of two generations from these parents were investigated. A raised mutation rate and physiological inferiority of the progeny was found, depending both on a dose and on the degree of oocyte maturity at the moment of the irradiation. The obtained data showed the instability of the irradiated genome in a number of generations.

Effects of power frequency alternating magnetic fields on reproduction and pre-natal development of mice.

Three groups of ICR male and female mice were exposed to 50-Hz, sinusoidal, alternating, horizontal magnetic fields of 0.0 mT (sham), 0.5 mT and 5.0 mT (rms) for 9 and 2 weeks prior to mating for males and females, respectively, through fertilization and until cesarean sectioning. Fetuses were collected by cesarean section on the 18th day of gestation. Approximately half were randomly selected for skeletal examination and the remainder used for visceral examination. No significant differences were found between the field- and the sham-exposed groups in pre-, post- and total implantation losses; number of live fetuses; sex ratio; live fetal weight; number of externally abnormal fetuses; and numbers of fetuses with skeletal and visceral anomalies. These results suggest that exposure to power-frequency magnetic fields has no major effects on reproduction and development in mice, and do not support the association of EMF exposure with adverse reproductive effects suggested by epidemiology.

Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis.

PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3sigma pathway.

Developmental abnormalities induced by X-irradiation in p53 deficient mice.

In order to assess the influence of p53 inactivation on radiation-induced developmental effects, male mice heterozygous for the wild-type p53 allele (mimicking the human Li-Fraumeni syndrome) were crossed with C57BL females, and their heterozygous p53+/- progeny were mated with each other to obtain p53+/-, p53-/- and p53+/+ embryos. Pregnant females were X-irradiated with 0.5 Gy on days 1 (pre-implantation period), 8 or 11 (organogenesis period) of gestation. Dissection of the pregnant females occurred on day 19 of gestation. The p53 genotype of the foetuses was determined by PCR from small pieces of soft tissues. Exencephaly was the only external malformation found in the control group. It affected essentially p53-/- female foetuses. A number of p53+/- and p53+/- control foetuses also showed dwarfism, or underdevelopment. In the group irradiated on day 1, the frequency of abnormal foetuses was, paradoxically, lower than that found in the control group. As in that group, exencephaly and dwarfism constituted the only anomalies that were found. Exencephaly affected only homozygous p53-/- females, while dwarfism concerned either p53-/- or p53+/- foetuses, with a majority of females. Irradiation on day 8 of gestation induced a significant increase in the frequency of abnormal foetuses, compared to the control group. Various malformations were observed in addition to exencephaly, including gastroschisis, polydactyly, cephalic oedema and cleft palate. All malformed foetuses were either homozygous p53-/- or heterozygous p53+/- while most affected foetuses were females, as was the case for dwarf individuals. Irradiation on day 11 did not cause an increase in the frequency of abnormal foetuses, in comparison with the controls. However, a large spectrum of external malformations was again noticed, as in the group irradiated on day 8. All affected foetuses were homozygous p53-/- and there were slightly more abnormal females than males (3 out of 5). No dwarfs were found in this group. Overall, these results confirm the importance of the p53 tumour-suppressor protein for normal embryonic development. They clearly show that homozygous p53-/- (or heterozygous p53+/- to a lesser extent) foetuses are more at risk for radiation-induction of external malformations during the organogenesis period, and that the risk of developing such malformations is much higher for females than for males. In contrast to results published very recently by others, we found that malformed foetuses resulting from an X-irradiation with a low-dose during the highly sensitive period of gastrulation are able to survive to birth.