Mojmír Mach

Evaluation of developmental neurotoxicity: some important issues focused on neurobehavioral development

Evaluation of developmental neurotoxicity: some important issues focused on neurobehavioral development Exposure of the developing organism to industrial chemicals and physical factors represents a serious risk factor for the development of neurobehavioral disorders, such as attention-deficit hyperactivity disorder, autism and mental retardation. Appropriate animal models are needed to test potentially harmful effects and mechanisms of developmental neurotoxicity of various chemical substances. However, there are significant human vs. rat differences in the brain developmental profile which should be taken into account in neurotoxicity studies. Subtle behavioral alterations are hard to detect by traditional developmental toxicity and teratogenicity studies, and in many cases they remain hidden. They can however be revealed by using special behavioral, endocrine and/or pharmacological challenges, such as repeated behavioral testing, exposure to single stressful stimulus or drugs. Further, current neurobehavioral test protocols recommend to test animals up to their adulthood. However some behavioral alterations, such as anxiety-like behavior or mental deficiency, may become manifest in later periods of development. Our experimental and scientific experiences are highly suggestive for a complex approach in testing potential developmental neurotoxicity. Strong emphasis should be given on repeated behavioral testing of animals up to senescence and on using proper pharmacological and/or stressful challenges.

Teratology – past, present and future

ABSTRACT Teratology is the science that studies the causes, mechanisms, and patterns of abnormal development. The authors present an updated overview of the most important milestones and stages of the development of modern teratology. Development of knowledge and society led to the recognition that causes of congenital developmental disorders (CDDs) might be caused by various mechanical effects, foetal diseases, and retarded or arrested development of the embryo and foetus. Based on the analysis of the historical development of hypotheses and theories representing a decisive contribution to this field, we present a survey of the six Wilson´s fundamental principles of teratology. The aim of observing these principles is to get insight into developmental relations and to understand mechanisms of action on the level of cell populations (elementary morphogenetic processes), tissues and organs. It is important to realise that any negative intervention into the normal course of these processes, either on genetic or non-genetic basis, inevitably leads to a sequence of subsequent changes resulting in CDDs. Moreover, the classical toxicologic monotonic doseresponse paradigm recently has been challenged by the so-called “low dose-hypothesis”, particularly in the case of endocrine active substances. These include some pesticides, dioxins, polychlorobiphenyls (PCBs), and bisphenol A. Despite modern approaches of molecular biology and genetics, along with top diagnostic techniques, we are still not able to identify the actual cause in more than 65 to 70% of all congenital defects classified as having an unknown etiology. Today CDDs include any birth defect, either morphological, biochemical, or behavioural.

Prenatal hypoxia in rats increased blood pressure and sympathetic drive of the adult offspring.

Decreased oxygenation during pregnancy and early periods of ontogeny can affect normal body development and result in diseases in adulthood. The aim of this study was to use the model of prenatal intermittent hypoxia (PIH) and evaluate the effects of short-term hypoxia at the end of gestation on blood pressure (BP) control in adulthood. Wistar rats were exposed daily to PIH for 4 h during gestational day 19 and 20. In adult male rats, heart rate (HR), systolic BP and pulse pressure (PP) were acquired by radiotelemetry during 1 week. On the basis of HR variability and BP variability, sympathovagal balance (LF/HF) and spontaneous baroreflex sensitivity (sBRS) were evaluated. Systolic BP and PP were significantly elevated in PIH rats in comparison with control rats during the light and dark phase of the day, while LF/HF increased only during the light phase of the day. In contrast, sBRS tended to decrease only during the dark phase in PIH rats. In all measured and calculated parameters, significant circadian rhythms were present and were not affected by PIH. In conclusion, our data suggest that short intermittent hypoxia at the end of gestation can increase BP and PP via significant changes in LF/HF, which occur especially during the passive phase of the day. Results suggest that minor changes in the autonomous nervous system activity induced by environmental conditions during the perinatal period may contribute to development of hypertension in adulthood.

Experimental modeling of hypoxia in pregnancy and early postnatal life

Experimental modeling of hypoxia in pregnancy and early postnatal life The important role of equilibrium of environmental factors during the embryo-fetal period is undisputable. Women of reproductive age are increasingly exposed to various environmental risk factors such as hypoxia, prenatal viral infections, use of drugs, smoking, complications of birth or stressful life events. These early hazards represent an important risk for structural and/or functional maldevelopment of the fetus and neonates. Impairment of oxygen/energy supply during the pre- and perinatal period may affect neuronal functions and induce cell death. Thus when death of the newborn is not occurring following intrauterine hypoxia, various neurological deficits, including hyperactivity, learning disabilities, mental retardation, epilepsy, cerebral palsy, dystonia etc., may develop both in humans and in experimental animals. In our animal studies we used several approaches for modeling hypoxia in rats during pregnancy and shortly after delivery, i.e. chronic intrauterine hypoxia induced by the antiepileptic drug phenytoin, neonatal anoxia by decreased oxygen saturation in 2-day-old pups. Using these models we were able to test potential protective properties of natural (vitamin E, melatonin) and synthetic (stobadine) compounds. Based on our results, stobadine was also able to reduce hypoxia-induced hyperactivity and the antioxidant capacity of stobadine exceeded that of vitamin E and melatonin, and contrary to vitamin E, stobadine had no adverse effects on developing fetus and offspring.

Early assessment of the severity of asphyxia in term newborns using parameters of blood count

Early assessment of the severity of asphyxia in term newborns using parameters of blood count Acute perinatal asphyxia is a major cause of death and neurological injury in newborn infants. Severe asphyxia can occur in infants around the time of birth for several reasons. The aim of our study was to find the most sensitive, easily obtainable and fast assessable parameter of the presence and quantification of asphyxia. In our study 39 term newborns (15 healthy term newborns and 24 asphyxial term newborns), from vaginal deliveries admitted within 24 hours of life were monitored and parameters of blood count from venous blood were assessed. Laboratory findings of blood count parameters revealed significant differences between term asphyxial and healthy newborns in erythrocyte count and hemoglobin and hematocrit values. Hematological changes observed early after delivery can determine the duration of hypoxemia (acute vs. chronic) and asphyxia of short duration may be accompanied without occurrence of polyglobulia.

Effect of prenatal administration of venlafaxine on postnatal development of rat offspring

Abstract About 3% of pregnant women are treated with antidepressant drugs during gestation. After delivery the number of treated women increases to 5 to 7%. Most prescribed antidepressants in pregnancy are selective serotonin re-uptake inhibitors and/or serotonin and noradrenaline re-uptake inhibitors, such as fluoxetine, paroxetine, sertraline, citalopram and venlafaxine (VENF). Despite the fact that VENF has been assigned to pregnancy category C by the FDA, experimental studies with this drug are rare. The aim of this pilot study was to investigate the effect of prenatal administration of VENF on early postnatal development of rat offspring and selected biochemical variables at weaning of pups. Pregnant female Wistar rats were treated with VENF from day 15 to 20 of gestation at the doses of 7.5, 37.5 and 70 mg/kg. Females were allowed to spontaneously deliver their pups. After delivery the pups were inspected for viability, gross malformation and they were weighed on day 0, 4 and 21 post partum. On day 21 post partum, the pups were killed, brains were removed from the skulls and blood samples were collected for biochemical assay (proteins, glucose-GOD, glucose-HEX, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and total antioxidant status). The study showed that prenatal VENF administration resulted in a mild maternal intoxication manifested by decreased body weight gain of pregnant females. There was no effect of the drug tested on the body and brain weights of offspring. No obvious morphological alterations were observed in the delivered pups. Similarly, there were no changes in the selected biochemical variables determined.

Subchronic perinatal asphyxia in rats: Embryo–foetal assessment of a new model of oxidative stress during critical period of development

Approximately 3% of annual births suffer from birth asphyxia and one million of these newborns die. The aim of this study was to develop a model for studying subchronic perinatal asphyxia (SPA) in rats. Pregnant animals were exposed to 10.5% O2 during sensitive stages of brain development for 4h a day. Biochemical variables were analysed immediately and 24h after asphyxia. SPA caused significant reduction of foetal weight, produced abnormalities of distal parts of the skeleton, and anomalies in the development of brain ventricles. Time-dependent changes were observed in several parameters indicating adjustment of the developing organism to the delivery. Whereas lactate was elevated immediately after asphyxia, glucose mirrored high energy needs 24h after the insult. Immunohistochemical examination of the placentas revealed overgrowth of acidic glycoconjugates in the extracellular matrix of vascular walls in the animals exposed to asphyxia. We observed the presence of muscle fibres in chorionic plate arteries and also in intraplacental arteries. The present model proved to be useful for the study of asphyxial conditions during pregnancy. As it is non-invasive and allows to control asphyxial conditions, it appears suitable for the screening and investigation of indicators of asphyxia in the mother and foetus.

Anxiolytic activity of pyridoindole derivatives SMe1EC2 and SMe1M2: behavioral analysis using rat model

Anxiolytic activity of pyridoindole derivatives SMe1EC2 and SMe1M2: behavioral analysis using rat model Anxiety and mood disorders have become very significant affections in the last decades. According to WHO at least one mental disease occurred per year in 27% of EU inhabitants (more than 82 mil. people). It is estimated that by 2020, depression will be the main cause of morbidity in the developed countries. These circumstances call for research for new prospective drugs with anxiolytic and antidepressive properties exhibiting no toxicity and withdrawal effect and possessing beneficial properties, like antioxidant and/or neuroprotective effects. The aim of this study was to obtain information about psychopharmacological properties of pyridoindole derivatives SMe1EC2 and SMe1M2, using non-invasive behavioral methods in rats. The battery of ethological tests (open field, elevated plus-maze, light/dark box exploration, forced swim test) was used to obtain information about anxiolytic and antidepressant activity of the pyridoindole derivatives. The substances were administered intraperitoneally 30 minutes before the tests at doses of 1, 10 and 25 mg/kg. In the behavioral tests, SMe1EC2 was found to exert anxiolytic activity in elevated plus maze with no affection of locomotor activity. The highest dose of SMe1M2 increased the time spent in the lit part of the Light/Dark box, however this result was influenced by inhibition of motor activity of the rats. Similar findings were observed also in elevated plus-maze, although these results were not statistically significant. In conclusion, from the results of our study it is evident that both pyridoindoles acted on the CNS. In the highest dose, SMe1M2 was found to possess rather sedative than anxiolytic or antidepressant activity.

Effect of prenatal phenytoin administration on the fine structure of rat myocardium and aorta.

Phenytoin (PHT) is an antiepileptic drug known to have teratogenic effects. The aim of this study was to examine the ultrastructure of the left ventricle, the left atrium, and the aorta of 3-month-old offspring and 4-month-old mother animals after oral PHT (150 mg/kg/day) administration to Wistar/DV rats on days 7-18 of gestation. Electron microscopy of the myocardium revealed a heterogeneous population of cardiomyocytes with conventional architecture, and hypoxia/ischemia-like subcellular changes. Cardiomyocytes of offspring hearts were more vulnerable to PHT administration compared with the mother animals. Atrial cardiomyocytes of both mother animals and offspring were less affected by PHT than the ventricular ones. In the myocardium, both interstitial fibrosis and injury of capillaries were noted. Electron microscopy of the aorta revealed a higher resistance of maternal endothelial and smooth muscle cells to PHT compared with offspring cells. Nuclei of endothelial and smooth muscle cells showed pronounced mitotic activity with one and/or two hyperactive nucleoli, more frequently observed in offspring. PHT administration resulted in aortic arteriogenesis in both offspring and mother animals. Interestingly, bundles of myocardial fibers consisting of ischemia-like altered cardiomyocytes with own capillary network were noted in off-spring aortic adventitia. These results are indicative of harmful effects of PHT on rat myocardium and aorta.

Toxicology as an academic discipline in European Universities.

The Federation of European Toxicologists and European Societies of Toxicology (EUROTOX) publishes this statement paper on the status of Toxicology as an academic discipline in Europe to draw attention to the critical importance of the discipline in protecting the health of consumers, workers and the environment. The discipline is essential in ensuring safety of food, drugs, pesticides and industrial products used in daily life. Due to reductions in the staffing of the discipline in many universities, EUROTOX needs urgently to remind governments and the public of the necessity to maintain and strengthen toxicology as an academic discipline in light of its essential role across an evergrowing range of activities. EUROTOX is the federation of national societies of toxicology in Europe with a combined membership of approximately 7000 with an additional 200 individual members. EUROTOX aims to foster toxicology, both scientifically and educationally, in all countries of Europe. We are concerned about the situation in academia which may impede the future development of the discipline. Across Europe, departments of Toxicology in universities have been reduced in size or even closed, posts have been not renewed, and currently heads of departments are being told of the intention to close their departments in the near future. Strengthening toxicology in academic institutions is a basic requirement for scientific innovation and for the education and development of young scientists. Toxicological expertise is instrumental in assessing and reducing human health risks in the work place and for consumers. In the public debate, toxicological expertise is also needed to evaluate evidence and to argue whether or not there is a scientifically founded reason for public health concerns. Governments across Europe need toxicology expertise to serve on committees to safeguard our future. This is a service to society and is an ongoing need which requires appropriately qualified individuals to maintain a safe environment for all. In the absence of appropriately experienced toxicologists, governments will be forced to seek toxicological input from non-experts who may inadvertently offer the wrong advice. This means we need to train toxicologists which means we need academic toxicology.