The role of Trp53 in the transcriptional response to ionizing radiation in the developing brain.

Brain formation results from a series of well-timed consecutive waves of cellular proliferation, migration and differentiation. Acute irradiation during pregnancy selectively interferes with these events to result in malformations such as microcephaly, reduced cortical thickness and mental retardation. In the present study we performed a straight-through cDNA-microarray analysis of the developing mouse brain at embryonic day E13, 3 h after in utero exposure to 50 cGy X-radiation. This dataset was used as an indication of genes involved in different pathways that are activated upon early radiation exposure, and for further evaluation using quantitative PCR (qPCR). Microarray and qPCR data revealed that the main activated pathways in irradiated wild-type embryos are involved in the regulation of a p53-mediated pathway that may lead to cell cycle delay/arrest and increased levels of apoptosis. To define whether the transcriptional radiation response was solely p53 mediated, we analysed the expression of cell cycle regulating genes in a Trp53 null mutant. The modulated expression of cell cycle regulating genes such as cyclins and Cdk genes indicated the induction of a cell cycle arrest, without evidence for the onset of apoptosis. Additional gene-expression studies have shown that various E2F transcription factors may be involved in this event. Together, these results provide a detailed view of the different p53-related mechanisms that are triggered in response to ionizing radiation in the developing brain.

Prenatal protracted irradiation at very low dose rate induces severe neuronal loss in rat hippocampus and cerebellum.

Prenatal irradiation is known to damage the developing brain. However, little is known about the consequences of very low dose rate prenatal protracted irradiation over several days on neuron numbers in the offspring brain, and on volumes of the corresponding brain regions. Pregnant Wistar rats were exposed either to a protracted gamma irradiation from embryonic day (E) 13 to E16 (0.7 mGy/min; total cumulative dose approximately 3 Gy) or were sham-irradiated. Thirty months old male and female offspring were then analyzed for alterations in hippocampal and cerebellar morphology. Using design-based stereology and the analysis of sets of sections systematically and randomly sampled to span the entire brain region of interest, a statistically significant decrease in numbers of hippocampal pyramidal and granule cells as well as of cerebellar Purkinje and granule cells (approximately 50%) was found in male and female irradiated offspring. The volumes of these brain regions were comparably altered. The analysis of only a "representative" section per animal yielded mostly non-significant trends. Evaluation of neuron densities showed no differences between prenatally irradiated and sham-irradiated offspring. Most importantly, very low dose rate prenatal protracted gamma irradiation did not result in the same morphologic alterations in the offspring brain as previously observed after prenatal single irradiation such as derangement of the laminar structure of pyramidal cells within the hippocampus or malformation of cerebellar lobules.

Genome organisation of the FBR-osteosarcoma virus complex: identification of a subgenomic fos-specific message.

The FBR murine virus complex together with the FBJ murine virus complex are known to be bone tumor inducers in newborn mice. Both transforming viruses have transduced c-proto-fos-derived sequences in their genome. FBR-MuSV was molecularly cloned as a biologically active 10-kbp EcoRI fragment from non-productively transformed rat embryo fibroblasts into Charon phage 4A (lambda MOL503) and subsequently subcloned in plasmid pBR322 (pMOL503). Its natural associated helper FBR-MuLV, excized as an internal 8.2-kbp PstI proviral DNA fragment from chronically infected NIH/3T3 cells, was cloned into the unique PstI site of pBR322. Comparative analysis of the restriction maps of FBR-MuSV and FBR-MuLV together with the electron microscopic analysis of heteroduplex DNA molecules formed between both molecular clones suggested that FBR-MuLV is the parental virus of FBR-MuSV. fos- and fox-specific DNA hybridisation probes identified a genomic sized 3.3-kb mRNA and a subgenomic 2.2-kb messenger RNA. Using a 5'-gag hybridisation probe, only the genomic 3.3-kb RNA molecule was detected, demonstrating that a donor splice site is present upstream of the gag sequences and used to generate the fos-specific 2.2-kb subgenomic mRNA.

Effect of X rays alone or combined with diethylnitrosamine on tumor induction in infant mouse liver.

The possible combined effects of the initiator diethylnitrosamine (DEN) with X rays on cancer induction in C57BL/Cnb mouse liver were evaluated. Four groups of infant mice were treated as follows: with DEN alone, with X rays alone, with DEN + X rays, and with X rays + DEN. Mice in each group were killed at 10-week intervals over 70 weeks. The following parameters were measured: body weight, liver weight, number and size of macroscopic liver lesions, and number and total surface of the different types of microscopic liver lesions. The number of induced liver foci and carcinomas was found to depend essentially on the dose of DEN. X irradiation did not produce any combined effect on the induction of foci and carcinomas when given 7 days before or after DEN administration.

Effect of neutrons alone or combined with diethylnitrosamine on tumor induction in the livers of infant C57BL mice.

The possible combined effects of the initiator diethylnitrosamine (DEN)+neutrons on the induction of foci, adenomas and carcinomas in the livers of C57BL/Cnb mice were evaluated. Four groups of infant mice were treated as follows: DEN alone, neutrons alone, DEN followed by neutrons and neutrons followed by DEN. Ten mice in each group were killed at 10-week intervals over 70 weeks. The following parameters were measured: body weight, liver weight, number and size of superficial macroscopic liver lesions, and number and total surface area of the different types of microscopic liver lesions. The rate of appearance of foci increased significantly at different times when a dose of 0.125 Gy of neutrons was administered 7 days before or after a dose of 1.25 micrograms of DEN. No significant differences were observed in the total surface area of foci and/or adenomas and carcinomas when increasing doses of neutrons were given 7 days before or after the administration of 1.25 and 2.5 micrograms of DEN.

Histone H1 kinase activity in one-cell mouse embryos blocked in the G2 phase by X-irradiation.

The activation of the p34cdc2/cyclin B complex is responsible for driving the cell cycle from the G2- to the M-phase. To investigate the effects of irradiation on the activity of the p34cdc2/cyclin B complex in preimplantation embryos, we irradiated one-cell mouse embryos with 2.5 Gy of X-rays at the early pronuclear stage, and measured the fluctuations of histone H1 kinase activity (a biochemical indicator of the kinase activity of the p34cdc2) at different times during the radiation-induced G2-arrest. BALB/c embryos were chosen for these experiments, since earlier results obtained in our laboratory had shown that such a treatment induces a G2-arrest of about 20 hours in more than 90% of the embryos. Our data showed that histone H1 kinase activity of irradiated embryos remained at a very low level during the period of G2-arrest. The level of activity found during late division of the G2-arrested embryos was also significantly lower in comparison with that of control embryos or irradiated embryos dividing without delay. All together, our results suggest that a) low levels of histone H1 kinase activity are sufficient for the division of one-cell embryos, b) there could be a link between the levels of histone H1 kinase activity in mitosis and the health status of the embryo.

Morphological effects of caffeine, okadaic acid and genistein in one-cell mouse embryos blocked in G2 by X-irradiation.

One-cell mouse embryos of the Balb/c strain normally divide at 18.5 h p.c. (post conception), but they suffer an extremely long G2 arrest when irradiated with 2 Gy X-rays 8 h p.c. at the early pronuclear stage. This could be an indirect effect of radiation on tyrosine dephosphorylation of the p34cdc2 subunit of a maturation or mitosis promoting factor (MPF), which normally occurs at the end of G2. This, in turn, would maintain MPF in an inactivated form and block entry into mitosis. Preliminary studies were undertaken at the morphological level to assess indirectly the validity of this hypothesis. For this purpose, irradiated and control embryos were exposed to different compounds, which are known to interfere, directly or indirectly, with the state of phosphorylation/dephosphorylation of p34cdc2. Caffeine (CAF; 2 mM) did not affect the time of first division of control embryos, but it completely suppressed the radiation-induced G2 arrest of embryos exposed to this compound from 17 h p.c., i.e. 1.5 h before the normal time of first cleavage. Under the same conditions, okadaic acid (OA; 3 microM), a specific inhibitor of phosphatases I and IIA, induced a rapid pronuclear membrane breakdown and a block of all control and irradiated embryos at metaphase. Genistein (GEN; 92 or 185 microM). A potent inhibitor of tyrosine kinases, increased the radiation-induced G2 arrest and even induced a dose-dependent G2 arrest in the control embryos. Embryos were exposed at different times following irradiation to a mixture of either CAF (2 or 5 mM) or OA (3 or 10 microM), and cycloheximide (CH; 5 micrograms/ml), a potent protein synthesis inhibitor. Reversion of G2-arrest by CAF was still seen in embryos exposed to CAF+CH from 17 h p.c. However, the proportion of irradiated embryos eventually able to cleave was lower than that obtained under the conditions of exposure to CAF alone. Embryos exposed to CAF+CH before 17 h p.c. were not able to cleave, regardless of the concentration of CAF used. Nuclear envelope breakdown still occurred in 100% control and irradiated embryos, following exposure to 3 microM OA+CH from 10 h p.c., or to 10 microM OA+CH from 8.5 p.c.(ABSTRACT TRUNCATED AT 400 WORDS)

Delayed effects of prenatal low-dose irradiation in the white matter of the rat brain.

PURPOSE: This study is part of a general search for the anatomical bases of the severe mental retardation syndrome caused by prenatal irradiation. More specifically, it seeks reasons for the high radiosensitivity of a white matter area, the cingulum of the corpus callosum. MATERIALS AND METHODS: Pregnant primiparous Wistar rats were exposed to X-rays at 12, 13, 14 or 15 days of gestation (E12, E13, E14 or E15) with single low doses of 180 mGy. A high dose of 500 mGy was given at E15 for a complementary study. The brains of the female offspring were collected at 1 and 3 months of age. The affinity of osmium tetroxide for the white matter was used to recognize and quantify cingulum areas in a series of coronal sections made at different positions along the antero-posterior axis of the brains. RESULTS: A 180 mGy dose of X-rays caused an atrophy of the cingulum; the effect was particularly significant in the 1-month-old brains after an exposure at E13 or at E14, and increased in the 3-month-old animals exposed at E15. The axonal size distribution was unchanged in the 3-month-old cingulums treated with 500 mGy at E15. CONCLUSIONS: The atrophy of the cingulum is due to a loss of axons, which are abundant in this area. As a consequence of axon loss, a reduction of the postnatal myelination enhances the volumetric decrease of the cingulum at 3 months of age.

Low-dose ionizing radiation exposure: understanding the risk for cellular transformation.

Radiation is energy transfer. When radiation has sufficient energy to remove an orbital electron from its atom, an ionized atom is formed, and radiation with the capacity to do this is called ionizing radiation. The primary effect of radiation is the induction of free radicals and Reactive Oxygen Species (ROS). All the molecules in every cell of the body are potential targets, but the final effect of radiation will be mainly of concern if the molecule impaired is a molecule critical for life. ROS are also generated as a result of the aerobic respiration (metabolic ROS) in much larger quantity than from the natural radiation background. During evolution, life has developed powerful control and repair mechanisms that greatly contribute to minimize the risks associated with the generation of free radicals and ROS. At low irradiation doses the probability of the risk is therefore proportional to the dose, and the ALARA (As Low As Reasonable Achievable) principle seems to be a valuable goal in radioprotection policies.

Embryonic death, dwarfism and fetal malformations after irradiation of embryos at the zygote stage: studies on two mouse strains.

Female mice of the BALB/c and CF1 strains were mated and irradiated with various doses of X-rays 7 h after presumed fertilization. 18 days later, females were killed and their uteri examined for prenatal mortality at the different stages of development. Living fetuses were weighed and examined for the presence of external malformations. A number of them were also examined for skeletal anomalies. Radiation induced mainly a dose-dependent increase of the preimplantation loss in the BALB/c strain and of the early postimplantation loss in the CF1 strain. Embryos of the BALB/c strain were refractory to the induction of teratogenic effects after such preimplantation irradiation. In CF1 mice, the frequency of malformed fetuses increased regularly after irradiation, the difference with controls being significant for the doses of 10, 50 and 100 cGy. Dwarfism occurrence also appeared to be increased by irradiation in this strain, although the importance of this effect varied depending on the criterion chosen for the assessment of dwarfs. With the definition proposed in the present paper, the increase in the frequency of dwarfs paralleled that of malformed fetuses, being significant after doses of 50 and 100 cGy. Irradiation did not increase the frequency of skeletal anomalies. A careful examination of the various data obtained to data led us to conclude that radiation may possibly be teratogenic in several mouse strains, when administered as early as during the one-cell stage and, to a lesser extent, during the following preimplantation stages. However, early prenatal mortality will remain by far the greatest risk associated with an exposure to radiation during this period. Moreover, the relativity of the risk of abnormality due to such irradiation should be considered in the context of the high prevalence of developmental defects spontaneously occurring during human pregnancy.

Cytogenetic effects of X-rays in the guinea pig female germ cells. I. The immature oocyte.

In female mammals, the immature oocyte is the germ cell most at risk, from the genetic point of view. We have previously shown that ovaries of newborn guinea pigs contain a great majority of immature oocytes and small numbers of maturing oocytes. At this time, all immature oocytes are in a typical diplotene stage, comparable to that of the human resting oocyte. Furthermore, the LD50 of the guinea pig oocytes is about 4 Gy, like that of human oocytes. Consequently, performing genetic studies on guinea pig oocytes irradiated just after birth constitutes a very useful approach to predict the mutational response of the human resting oocyte. Female guinea pigs were X-irradiated with 1 or 2 Gy within 24 h of birth, and their meiotically competent oocytes were collected and cultured to the metaphase I stage, 1 year after treatment, ensuring that all oocytes were at the immature stage at the time of irradiation. The proportion of oocytes that showed chromosome aberrations (breaks, fragments or interchanges) was only 1-2% Gy(-1). Although not definitive, these results provide support for a low risk in women from an exposure of the immature oocyte to low doses of radiation.

Cytogenetic effects of X-rays in the guinea pig female germ cells. II. The maturing oocyte.

In a previous study, we showed that the guinea pig constitutes one of the best models to evaluate the genetic risk associated with an irradiation of the human female germ cells. Herewith, experiments were undertaken to evaluate the chromosomal radiosensitivity of oocytes of this species at two different stages of follicular development, separated by only 1 week. Female guinea pigs were X-irradiated on the ovaries, at either the beginning (day 3) or the middle (day 10) of the 17-day oestrous cycle. The doses delivered were 1 or 2 Gy. Meiotically competent oocytes were collected 1 week after irradiation (day 3) or immediately thereafter (day 10), and they were cultured to the metaphase of the first meiosis (MI) and examined for the presence of chromosome aberrations. Our data demonstrated a dramatic increase in the radiosensitivity of the oocyte during this short time interval: oocytes irradiated at the beginning of the oestrous cycle had a low frequency of chromosome aberrations, while those irradiated at the middle of the oestrous cycle (when growing Graafian follicles are clearly visible at the surface of the ovaries) exhibited heavy chromosome damage. However, we also found that oocytes irradiated at the middle of the oestrous cycle were eliminated from the ovaries in a few days, after their evolution to the MII stage. The stimulation of the first meiotic division by radiation required less than 24 h after doses of 1 or 2 Gy and was probably due to a rapid atresia of the large follicles containing the oocytes. On the basis of these results, it can be concluded that the radiosensitivity of the nearly mature guinea pig oocyte (1 week before ovulation) is clearly much higher than that of the corresponding stage in the mouse, both in terms of sensitivity to killing and to induction of chromosome aberrations.

Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests.

The mutagenicity of diphenylhydantoin (DPH) and its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), has been re-evaluated by the Ames test using Salmonella typhimurium and, for DPH only, by an in vitro cytogenetic test with human lymphocytes and a turbidimetric assay of tubulin polymerization. As negative results were obtained in all test systems used here, one has to conclude that DPH is devoid of mutagenic properties.

Radiation-induced chromosome aberrations in guinea-pig growing oocytes, and their relation to follicular atresia.

The female guinea-pig has been shown to represent a good model to investigate the genetic hazard of ionizing radiation in humans. The sensitivity of the guinea-pig oocytes to radiation-induced chromosome aberrations was, therefore, studied at different stages of oocyte and follicular growth. The sensitivity of oocytes enclosed in small follicles (15 weeks before ovulation) was found to be low and comparable to that of immature oocytes present at birth. The sensitivity of growing oocytes remained low and almost constant until 3 weeks before ovulation, from which time it began to increase. The most dramatic increase of sensitivity occurred during the last week preceding ovulation: about 90% of oocytes X-irradiated with 4Gy, 2 days before ovulation showed one or more chromatid interchanges, as compared to 20% for those irradiated with the same dose 1 week earlier. A comparison of our results with those found by others in the mouse shows that considerable differences of sensitivity exist between oocytes of these two species irradiated at similar stages of development. The possible reasons for these differences are discussed.

Evaluation of the ability of paracetamol to produce chromosome aberrations in man.

The ability of paracetamol to induce structural chromosome aberrations in human peripheral blood lymphocytes in vivo was evaluated in volunteers who had been administered a single oral dose of 3 g paracetamol, in patients who had received 2 g of propacetamol by intravenous infusion every 6 h for at least 7 days, and in self-poisoned patients who, for suicidal reasons, had ingested more than 15 g paracetamol. In addition to the in vivo observations, the effectiveness of paracetamol to interfere with fusorial microtubule polymerisation was assayed in vitro in order to detect a possible effect of paracetamol on the distribution of chromosomes during cell division. The negative results obtained in all those assays strongly suggest that paracetamol has no mutagenic properties in human. There was, indeed, no significant difference in the percentage of abnormal cells before and after application of paracetamol in volunteers (0.2% before ingestion of 3 g paracetamol, 0.12% after 24 h, 0.04% after 72 h and 0.04% after 168 h) and in patients (0.5% of abnormal cells before treatment versus 0.44% after intravenous infusion of a total of 28 g paracetamol). Moreover, the yield of abnormal cells was not modified in self-poisoned persons (0.24%), in spite of an important decrease in the mitotic index of the PHA stimulated lymphocytes. In the in vitro assay, no inhibition of microtubule polymerisation was detected with concentrations of 2.5, 5 and 10 mM paracetamol.

The microcirculation of bone and marrow in the diaphysis of the rat hemopoietic long bones.

The nature of the microcirculation of the diaphyseal portion of long bones and the adjacent bone marrow is poorly understood. The purpose of this study was to describe the blood supply in the diaphyseal cortex and the relationship of the bone vascular circulation to that of the bone marrow in the growing rat. India ink-gelatin was infused in the arterial system of 3-month-old rats and the vascularization was determined from histological sections. In some studies the periosteal circulation was blocked but the nutrient and metaphyseal arteriole systems were left intact. In the growing rat, most of the vascular flow appears to be centripetally through the diaphyseal cortex and this appears to be the primary blood supply for the adjacent bone marrow. The India ink traversed the cortex and entered the marrow through osteal canals at the endocortical surface. At the marrow-endocortical bone surface interface, ink exiting from the osteal canals filled the adjacent marrow sinusoids in what appeared as "bush-like" structures. From the bone marrow the ink appeared to drain into the central vein. Some arterioles from the nutrient system were found to penetrate the inner two thirds of the cortical bone and then re-enter the bone marrow. The centripetal flow of blood and the importance of the cortical flow for perfusion of the hemopoietic tissue was further documented when periosteal flow was obstructed. In this situation, the cortical bone and adjacent bone marrow were not perfused while the nutrient system and central vein were filled with ink.(ABSTRACT TRUNCATED AT 250 WORDS)

[Inhibition by methyl mercury chloride and mercuric chloride of the in vitro polymerization of microtubules]. / Inhibition par le chlorure de méthyl mercure et le chlorure de mercure de la polymérisation in vitro des microtubules.

Mercury was tested at the same concentration but under two different forms, organic CH3HgCl and inorganic HgCl2, in order to compare its relative inhibitory effect on in vitro microtubules polymerization. Induced by GTP and glycerol 8 M, tubulin polymerization was completely inhibited by HgCl2 10(-3) M while a 75.8% inhibition was measured for CH3HgCl2 10(-3) M.

[Lead toxicity in mice embryos]. / Toxicité du plomb pour les embryons de la Souris.

Administration to female mice before coïtus and to pregnant female mice of an alimentation containing 0.1 p. 100 lead acetate imparied the fertility. Studies on the changes of the ultrastructure of the embryos during the first stages of development do not allow to detect lesions unless on day 7 where lead inclusions are detected in the mitochondria.

The ultrastructure of the lung after exposure to ionizing radiation as seen by transmission and scanning electron microscopy.

Male mice of the BALB/c strain were exposed at an age of 12-14 weeks, to different doses of X-rays, either to the entire thorax or to the right hemithorax. At various times after exposure mice were sacrificed and the lung was examined by transmission or scanning electron microscopy. Radiation symptoms following exposure to 15-20 Gy can be divided into three phases: Early, from a few hours to a few weeks after exposure, changes in capillary permeability and morphological alterations in all types of lung cells are prominent. At intermediate times, from a few weeks to about 7 months after exposure, the symptoms of radiation pneumonitis arise essentially, as a consequence of the action of radiation on the epithelium of the alveoli, and are characterized by a large increase in size, and probably in number of type II epithelial cells. Scanning electron microscopy brings additional arguments that the lesions in the epithelial cells, mainly of type II, are the principal cause of radiation pneumonitis and that capillary damage probably plays a secondary role.