Impact of asphyxia on red blood cell folate concentration levels in newborns.

OBJECTIVES: To determine whether red blood cell (RBC) folate concentration levels are correlated with the occurrence of neonatal asphyxia and to study the effects of gestational age, gender, and mode of delivery on RBC folate concentration levels in newborns. BACKGROUND: Asphyxia is one of the frequent causes of morbidity and mortality of newborns. Severe perinatal asphyxia can arise due to many factors. METHODS: In a prospective study, the RBC folate concentrations were determined on day 1 of life in the whole group (n=181) of full-term (n=121) and preterm (n=60) newborns. Immunochemical analysis for the determination of folate in erythrocytes was performed. RESULTS: RBC folate concentration levels in asphyxiated newborns (n=16) were significantly decreased (median 974 ng/ml; p=0.023) in comparison with healthy newborns. On the other hand, the RBC folate concentration levels were significantly increased in preterm newborns (median 1,212 ng/ml; p=0.01) in comparison with full-term newborns (median 1,098 ng/ml). Higher RBC folate concentration levels were found in newborns which had been delivered by Caesarean section (median 1,188 ng/ml; p=0.02) compared to those born vaginally (median 1,098 ng/ml). CONCLUSION: Our results confirmed a significant decrease in RBC folate concentration in asphyxiated newborns on their first day of life (Fig. 4, Ref. 36).

Effects of combined exposure to pyridostigmine bromide and shaker stress on acoustic startle response, pre-pulse inhibition and open field behavior in mice.

The present study investigated the effect of combined exposure of pyridostigmine bromide (PB) and chronic shaker stress on acoustic startle responses (ASR), pre-pulse inhibition (PPI) and open field behavior of adult C57BL/6J mice. PB (10 mg kg(-1) day(-1) for 7 days) or saline was administered subcutaneously using osmotic Alzet minipumps implanted under the skin on the back of the mice. At the same time, the mice were exposed to 7 days of intermittent shaker stress. They were tested for ASR (100 dB and 120 dB stimuli) and PPI (70 dB + 100 dB and 70 dB + 120 dB) in the acoustic startle monitor system. The mice were assessed during the shaker stress on days 2 and 7 and 7, 14, 21 and 28 days after discontinuation of treatment. Separate groups of mice were tested in the open field in 15 min sessions on days 1, 3 and 6 during shaker stress and PB treatment. Exposure of mice to PB resulted in an exaggerated ASR, reduced PPI and non-significant decrease in locomotor activity. These behavioral changes were apparent only during exposure to PB. Repeated shaker stress did not have any effect on sensorimotor functions or open field behavior of mice. There was no prolonged or delayed effect of PB and/or stress on individual behavioral variables. The study found C57BL/6J mice to be behaviorally sensitive to PB treatment.

Teratological study of the herbicide 4-chloro-2-methylphenoxyacetic acid in rabbits.

4-Chloro-2-methylphenoxyacetic acid (MCPA) is an aryloxyacetic acid derivative categorised as a plant hormone herbicide. The aim of the study was to evaluate the effect of MCPA on pregnant females and the prenatal development of rabbits. The substance tested was administered orally to pregnant New Zealand White rabbits from day 6 to day 27 of gestation at doses of 5, 10 and 25 mg kg(-1) day(-1). The animals were killed on day 28 of gestation and live fetuses were examined for gross, skeletal and visceral anomalies. Administration of MPCA did not induce any signs of maternal toxicity. There was a significant decrease of fetal and placental weight compared with controls at the highest dose of MPCA. No adverse effect of the substance tested was seen on uterine content variables, e.g. corpora lutea, pre-implantation and post-implantation loss, early, late resorptions, live and dead fetuses and sex ratio. Rabbit fetuses treated with the middle and highest doses of MPCA had a significantly elevated incidence of skull and pelvic bone delays. In conclusion, prenatal administration of MCPA did not exhibit a teratogenic effect on rabbit fetus development.

Protection of newborn organism against effect of oxidative stress.

OBJECTIVE: The aim of the present study was to investigate influence of oxidative stress on newborn organism, in terms of the dynamics of malondialdehyde (MDA) concentration changes and of the activities of selected antioxidants in asphyxial newborns on the 1st and 5th day of life. METHOD: In the group of 62 preterm and term asphyxial newborns, characterized by the presence of asphyxial criteria and admitted within 24 hours of life, the plasma concentration of MDA and level of total antioxidant status (TAS) were followed. RESULTS: Dynamic changes of MDA signalized active process of lipoperoxidation (LP) and values of TAS were decreased in comparison with the capacity of adult patients.

Effect of melatonin on neurobehavioral dysfunctions induced by intrauterine hypoxia in rats.

Intrauterine hypoxia associated with oxidative stress represents an important risk factor for development of neurobehavioral dysfunctions. In the present study, we investigated the potential protective effect of melatonin (MEL) on neurobehavioral dysfunctions induced by chronic intrauterine hypoxia in rats by the anticonvulsant drug phenytoin (PHT), which is known by its teratogenic potential. Pregnant female rats (Wistar/DV) were orally treated by PHT (150 mg/kg) from day 7 to 18 of gestation. MEL was dissolved in drinking water (40 microg/ml) and administered from day 0 to 19 of gestation. Neurobehavioral development of offspring was evaluated from birth up to day 90 of postnatal life. The results of the study confirmed the high behavior-teratogenic potential of PHT. Prenatal administration of PHT resulted in delayed neuromotor and reflex development, decreased exploration in the open field, abnormal "circling" and impaired performaces in water maze. Co-administration of MEL failed to have any effect on neurobehavioral dysfunctions induced by PHT treatment. Even administration of MEL alone caused developmental alterations in offspring manifested by accelerated testes descent and delayed onset of negative geotaxia and startle reflex. The results suggest to pay increased attention to MEL concerning its exogenous use during pregnancy.

Effect of melatonin on biochemical variables induced by phenytoin in organs of mothers, foetuses and offsprings of rats.

The present pre- and postnatal study was carried out to investigate the effect of melatonin (MEL), a potent antioxidant, on biochemical variables in the in vivo model of intrauterine hypoxia in rats. Chronic hypoxia was induced pharmacologically by the administration of the anticonvulsant phenytoin (PHT) during pregnancy. Rats were orally treated by PHT (150 mg/kg) from day 7 to 18 of gestation. MEL in drinking water (40 microg/ml) was administered from day 0 to 19 of gestation. The activity of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAGA) and the level of glutathione (GSH) were used as markers of tissue damage. In the prenatal study PHT-induced toxic damage was associated with an increase in NAGA activity and decrease of GSH level in placenta and in maternal serum and heart. MEL partially inhibited the changes of NAGA activity given above. MEL was able to increase only the decreased level of GSH in maternal heart. PHT decreased the level of GSH and increased the activity of NAGA in foetal organs, the improvement occurred in the liver and lungs, but not in foetal brain. In the postnatal study a significant increase of liver GSH level was found in all (control, MEL, PHT, MEL+PHT) groups of 1-day-old pups, while the activity of NAGA remained unchanged. We did not observe any significant differences in NAGA activity in the lungs and heart of pups. MEL increased the GSH level in lungs and heart. We concluded that administration of MEL during pregnancy partially inhibited the biochemical changes induced by PHT.

Effect of melatonin and stobadine on maternal and embryofoetal toxicity in rats due to intrauterine hypoxia induced by phenytoin administration.

The aim of the present study was to test the hypothesis that the natural antioxidant melatonin (MEL) and the synthetic antioxidant stobadine (STO) could reduce the incidence of maternal and embryofoetal toxicity in rats due to intrauterine hypoxia. Chronic hypoxia was induced pharmacologically by the administration of the anticonvulsant phenytoin (PHT) during the entire period of pregnancy. PHT disturbed the normal course of pregnancy, affected reproductive parameters and increased the incidence of skeletal anomalies. MEL did not protect the PHT-induced development toxicity in rat. On the other hand, STO partially prevented PHT-induced reduction of foetal and placental weights. Administration of STO also decreased the frequency of pre- and post-implantation loss and resorptions in the PHT group. We concluded that pretreatment of pregnant rats with STO prevented to a certain extent reproductive and foetal development alterations caused by chronic intrauterine hypoxia.

Determination of selective biochemical variables in pregnant and lactating mice after stobadine administration.

The aim of this study was to investigate the effects of the oral administration of stobadine (STB), a neuro- and cardioprotective drug with high antioxidant properties, on selective biochemical variables in pregnant and lactating mice. STB was administered orally at a dose of 50 mg/kg from day 15 of gestation to day 21 of lactation. Creatinine and urea were determined in serum, while acidity, proteins, glucose, ketones, bilirubin, urobilinogen, blood and creatinine were determined in urine from females on days 0, 15 and 18 of gestation and on days 7, 14, 21, and 28 postpartum (pp). In the biochemical variables investigated, no significant differences in STB-treated animals compared with controls were recorded on any of the days studied. Histopathological examination of kidney tissue did not reveal any adverse effect of STB administration.

Effect of repeated administration of the antioxidant stobadine on the behavior of singly-housed male mice in intraspecies conflict.

The aim of the present study was to assess the effect of repeated oral administration of stobadine (70 mg/kg) on the occurrence of selected behavioral elements during exposure to an intraspecies conflict between singly-housed and group-housed male mice. Isolation induced timidity (defensive-escape behavior without attacks) in most mice (87%). This isolation-induced timidity was reduced after stobadine treatment. In the stobadine-treated group, sociable activities (following, climbing) were also decreased. After discontinuation of the treatment (18 days), aggressive behavior tended to increase in the stobadine-treated group. The results of this study are indicative of an inhibitory effect of repeated administration of stobadine on some behavioral activities of singly-housed male mice in an intraspecies conflict.

Placental transfer of the antioxidant stobadine at different gestational stages in rabbits.

The distribution of [3H]-stobadine, a pyridoindole antioxidant, was investigated in New Zealand white rabbits and their fetuses on days 20 and 27 of gestation. The concentrations of [3H]-stobadine were determined in maternal and fetal organs after oral administration in a single dose of 5.0 mg/kg. The results of the study showed that during the late period of gestation the fetal organs, especially the brain and heart, were under the protective action of the antioxidant stobadine.

Neonatal stress alters habituation of exploratory behavior in adult male but not female rats.

The effect of monosodium-L-glutamate (MSG) administration in the neonatal period on habituation of exploratory behavior related to gender differences was investigated. Rats of both sexes were intraperitoneally treated with MSG (4 mg/g) or hypertonic saline (10% NaCl) on postnatal days 2, 4, 6, 8, and 10. On postnatal day 65, the animals were tested in an open-field test during 4 consecutive days, once daily in 6-min sessions. The rapidity of habituation of exploratory behavior during repeated exposure to the open field (interrupted habituation) and over individual sessions (uninterrupted habituation) was evaluated by using the method of linear regression. Compared to intact controls, there were no significant differences found in interrupted habituation, neither in males nor in females. Uninterrupted habituation in neonatally treated males was slowed down in the first 2 days of testing. No differences in adult behavior between treated groups (MSG and hypertonic saline) were observed, i.e., there were no late effects specific for neonatal MSG administration. In females, uninterrupted habituation was not affected. Males proved to be more sensitive to neonatal stress associated with injections of MSG or hypertonic saline than females, and showed feminine-like habituation in the new environment.

Evaluation of long-term administration of the antioxidant stobadine on exploratory behaviour in rats of both genders.

Stobadine (STO) is a prospective neuro- and cardioprotective drug with high antioxidative properties. The aim of this study was to ascertain the effect of long-term administration of STO on exploratory behaviour and habituation processes in adult virgin female and male rats. Stobadine was administered by oral gavage in a single dose of 50 mg kg(-1) day(-1) for a total of 56 days. The animals were tested for exploratory behaviour-intensity of motor and vertical activity in an open field test in three blocks of measurements (initial screening; after 56 days of STO administration; and 28 days after the last treatment). The rate of decline of motor activity was evaluated during four consecutive days of testing (interrupted habituation). Administration of STO resulted in transient inhibition of exploratory behaviour in female rats without overtly detectable toxicity. Exploratory behaviour of males was not affected by STO treatment.

Placental transfer of stobadine in rabbits.

Stobadine, a pyridoindole antioxidant, was investigated for its placental transfer and distribution in New Zealand white rabbits on the 27th day of gestation. The concentrations of stobadine were determined in maternal and foetal organs (plasma, brain, heart) at 30, 60, 120, and 360 minutes after oral administration of the drug in a dose of 5 mg/kg. The results obtained proved that after oral stobadine intake by rabbits at the stage of advanced pregnancy both maternal and foetal organs were under a certain drug level which could act protectively against oxidative stress--frequently occurring during late organogenesis, foetal stages and delivery, as well as during early postnatal development.

Antioxidant stobadine and neurobehavioural development of the rat offspring.

Stobadine (STO) is a potential neuro- and cardioprotective drug with high antioxidative properties. The presented study investigated the effects of oral STO administration (5, 15 and 50 mg/kg/d) during pregnancy and lactation to dams on neurobehavioural development of their offspring (body growth and maturation, sensory functions, neuromotor and reflex development, levels of activity and emotional reactivity, memory and learning processes). The results of our experiments showed that long-term administration of STO had no adverse effects on the course of pregnancy and lactation in dams and on the neurobehavioural development of offspring.

Toxicological evaluation of the new calcium antagonist VULM 993.

The tolerance of the new calcium antagonist VULM 993 was investigated in a series of toxicological studies. The following results were obtained: the maximum tolerated oral dose in acute toxicity was 10,000 mg/kg for mice and 6600 mg/kg for rats, for venous administration it was 26.1 mg/kg in mice and 32.2 mg/kg in rats. In subacute oral toxicity test in rats, VULM 993 showed no toxic effect up to 300 mg/kg/d. The drug was not teratogenic in rats (5, 50 or 250 mg/kg/d, p.o.). VULM 993 did not show any positive response in tests for genotoxicity in vitro. Transplacental study of VULM 993 in rabbits indicated active placental barrier function in the late stage of pregnancy. The toxicological profile of VULM 993 is characterised by a high tolerance in all relevant species of experimental animals, and no biologically significant mutagenic potential was recorded.

Protective effect of the antioxidant stobadine against cyclophosphamide and irradiation induced oxidative stress.

The antioxidant stobadine was tested for its efficiency against oxidative stress in model experiments with ICR nonpregnant mice exposed either to cyclophosphamide (80 mg/kg) or whole body 60Co (6.5 Gy) irradiation. In a teratological experiment, pregnant mice were exposed to cyclophosphamide (10 mg/kg) from day 11 to 17 of gestation. Toxicity was measured by determining the lysosomal enzymes acid phosphatase and N-acetyl-beta-D-glucosaminidase. Cyclophosphamide and irradiation caused a significant increase in acid phosphatase and N-acetyl-beta-D-glucosaminidase activity in the spleen of nonpregnant mice. In the liver, lysosomal enzyme activities were unchanged and no changes in protein levels were recorded. In pregnant mice, acid phosphatase and N-acetyl-beta-D-glucosaminidase activities were increased in the spleen. An increase in foetal acid phosphatase liver activity was found. Pretreatment with stobadine prior to cyclophosphamide and irradiation significantly diminished the biochemical changes in both nonpregnant and pregnant mice. We conclude that stobadine is able to protect mice against cyclophosphamide- or irradiation-induced oxidative stress.

Teratological study of the antioxidant stobadine in rats.

The potential teratogenicity of the antioxidant stobadine (STO) was studied in Wistar rats. Daily oral doses of 5, 15 and 50 mg/kg STO were given from the 6th day of gestation up to weaning of pups--day 21 post partum. No significant differences between the STO treated groups and the control group were found in litter size, pre- and postimplantation losses and foetal body weight. External, skeletal and internal examinations of the foetuses revealed no evidence of teratogenesis. The offspring from the STO treated dams exhibited a high survival rate in their postnatal development. It can be concluded that STO had no adverse effects on the pre- and postnatal development of the offspring in rats.

Changes of exploratory behaviour and its habituation in rats neonatally treated with monosodium glutamate.

In an attempt to elucidate mechanisms involved in adaptation to a novel environment, consequences of neurotoxic damage induced by administration of monosodium glutamate (MSG) to both male (n = 42) and female (n = 45) rats in the early postnatal period were studied. Rats treated with MSG and appropriate controls were tested on postnatal days 21 and 65 for alterations of exploration and the rapidity of habituation changes in an open field test. Compared with intact animals, a high dose of MSG (4 mg/g) increased exploratory behaviour, with a subsequent decrease in the rapidity of habituation of male rats. Neonatal stress represented by hypertonic saline injection in a vehicle-control group induced a slight increase of exploratory behaviour as compared with intact animals. Males proved to be more vulnerable to neonatal MSG treatment and handling than females. These results suggest a negative effect of neonatal stress and treatment with MSG on habituation to a new environment in male rats.

Effect of long-term administration of stobadine on exploratory behaviour and on striatal levels of dopamine and serotonin in rats and their offspring.

Stobadine (STB), a cardioprotective drug, was evaluated for its effect on the intensity and habituation of exploratory behaviour in open field testing and on the levels of striatal dopamine (DA), serotonin (5-HT) and their metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyindole-3-acetic acid) in rats and their offspring. Dams were treated by oral gavage with STB (50 mg kg-1) for a total of 56 days from 14 days before mating to day 21 postpartum (pp). The first open field measurements of the dams were performed over 4 days at the beginning of the experiment, the second on days 21-24 pp and the third on days 49-52 pp (recovery period). Their offspring were tested on postnatal (pn) days 30-33 and 60-63. The biochemical analysis (HPLC with electrochemical detection) in the dams was performed at the same time schedule as given for the open field testing, but in their offspring only on pn day 60. Motor activity of the dams was decreased on days 21-24 pp. The increase of motor activity in female offspring was observed on pn days 30-33. Neurochemical analysis of the striatum of the dams revealed a significant increase of the levels of DA, 5-HT and 5-hydroxyindole-3-acetic acid. In male offspring the levels of DA were significantly decreased, whereas in females the levels were increased. These results suggest that maternal administration of STB resulted both in dams and their offspring in minor alterations in spontaneous behaviour components and changes in the dopaminergic and serotonergic system, but without inducing overtly detectable toxicity.

Effect of long-term administration of stobadine to rats on selective variables of spontaneous behaviour of their offspring.

The present study investigated the effect of long-term administration of the cardioprotective drug stobadine (STB) to dams on selective variables of spontaneous behaviour of their offspring in open field (horizontal and vertical activities, frequency and duration of grooming, and duration of total activity and immobility) tested on day 60 of age. The treatment of dams with STB significantly increased horizontal activity of offspring in both sexes. The other variables studied were not affected, with the exception of a significant increase in the frequency and duration of grooming and in the duration of total activity in females compared to males from STB treated dams.