Oxidative damage in chemical teratogenesis

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.     

Modulation of embryonic glutathione peroxidase activity and phenytoin teratogenicity by dietary deprivation of selenium in CD-1 mice

Selenium (Se)-dependent and -independent glutathione (GSH) peroxidases detoxify H2O2 and lipid hydroperoxides, which may mediate the teratogenicity of phenytoin and related xenobiotics. To test this hypothesis, CD-1 mice were placed on Se-deficient diets for 15, 25 or 40 days and bred so that the day of analysis corresponded to gestational day 11. In Se-replete control animals, embryonic peroxidase activities were only 5% of activities in maternal liver (P < .05). After 15 days of Se deprivation, maternal activities for H2O2 (reflecting Se-dependent peroxidase) and cumene hydroperoxide (CmOOH) (reflecting both Se-dependent and -independent peroxidases) were reduced to 20% (P < .05) and 35% of controls, respectively. At this time, the incidence of fetal cleft palates initiated by phenytoin (55 mg/kg intraperitoneally on gestational days 11, 12 and 13) was doubled, from 12% to 25% (P < .05). Selenite rescue (Na2SeO3, 350 micrograms/kg intraperitoneally on day 9) restored maternal and embryonic peroxidase activities and completely inhibited phenytoin-initiated postpartum lethality and fetal resorptions in animals that had been Se depleted for 15 days. After 40 days of Se deprivation, maternal and embryonic peroxidase/H2O2 activities were reduced to < 1% and 27% of Se-replete controls, respectively. In contrast, maternal peroxidase/CmOOH activity was increased to 70% of controls, reflecting induction of Se-independent peroxidase, compared with that with 15 days' depletion. Phenytoin-initiated cleft palates with 40 days' depletion appeared to be reduced (16%) compared with Se-replete controls (24%) (P < .07). In 40-day Se-depleted animals given selenite rescue, the 10% incidence of cleft palates was significantly lower than that in the 40-day Se-replete group (24%) but not the Se-depleted group (16%). This is the first demonstration of reduced Se-dependent GSH peroxidase activities in embryonic tissues with dietary Se-deprivation. The results implicate reactive oxygen species and lipid hydroperoxides in the mechanism of phenytoin teratogenicity and suggest that GSH peroxidases are important embryoprotective enzymes.     

Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro

Isotretinoin (ITR), a teratogen in many species, is associated with increased oxidative stress. Metallothionein (MT) is an important tissue antioxidant whose concentrations are induced by zinc. To study the role of supplemental Zn as an inducer of embryonic MT, we injected pregnant CD-1 mice subcutaneously with saline vehicle, or 20 or 40 mg/kg Zn on gestational day (GD) 6.5. After 48 h, embryonic MT concentrations increased in a dose-related manner (r = 0.64, P < 0.05) with Zn treatment. The possible protective role of Zn pretreatment against ITR teratogenicity was investigated in vivo and in vitro. CD-1 mice were pretreated with saline or Zn (20 and 40 mg/kg) on GD 8.5 and 9.5. ITR was administered to both groups of mice via three intragastric intubations of 100 mg ITR/kg at 4 h intervals on GD 10.5. On GD 18.5, Zn pre-treated mice demonstrated decreased ITR-mediated growth retardation, cleft palates and postpartum mortality. A reduction in embryonic MT concentrations was observed in mice exposed to ITR. Mouse embryos cultured on GD 8.5 with an addition of 15 micromol/L Zn for 48 h had a sixfold greater MT concentration (688 microg/g protein) than controls. The Zn pretreatment of cultured embryos prevented malformations and lessened growth retardation caused by 24 h exposure to 17 micromol/L ITR. These results suggest that Zn-mediated induction of MT in mouse embryos could protect against ITR teratogenicity.     

Mucogingival orthodontic and periodontal problems

Di-n-butyltin diacetate (DBTA) has been shown previously to cause malformations such as cleft mandible, ankyloglossia and fused ribs in rat foetuses after oral treatment of dams on day 8 of gestation. In this study, effects of pretreatment with carbon tetrachloride (CCl4) on the teratogenic activity of DBTA were examined in Wistar rats. Pregnant rats were pretreated sc with CCl4 (0.5 ml/kg) on days 6 and 7 of gestation and were treated orally with DBTA on day 8 of gestation at doses of 0, 4.5, 10 or 22 mg/kg. Caesarean sections were performed on day 20 of gestation, and foetuses were examined for external and skeletal anomalies. Pretreatment with CCl4 enhanced the incidence of external and skeletal malformations caused by DBTA. Concentrations of di-n-butyltin in embryos, maternal liver and maternal blood on day 9 of gestation were increased by CCl4 pretreatment, which inhibited the activity of maternal hepatic microsomal drug-metabolizing enzymes. These results suggest that there is little probability that metabolites of DBTA make a critical contribution to the teratogenicity of DBTA.     

Effect of free radical scavengers on diaphragmatic fatigue

Recent studies have suggested that free radical scavenger administration reduces the rate of development of diaphragm fatigue. Much of this work has been done, however, using in vitro muscle preparations; the purpose of the present study was to assess the effect of scavengers on in vivo diaphragm contractile function. To accomplish this, we compared the rate of development of fatigue of the electrically stimulated diaphragm in four groups of dogs: (1) animals given intravenous polyethylene glycol adsorbed superoxide dismutase (PEG-SOD, 2,000 units/kg) 1 h before a fatigue trial; (2) a group given intravenous dimethylsulfoxide (DMSO, 0.5 ml/kg of a 50% solution) before fatigue; (3) a group given saline before fatigue; and (4) a group treated with denatured PEG-SOD (2,000 units/kg) before fatigue. We measured diaphragmatic concentrations of thiobarbituric acid reactive substances (TBAR), a marker of free radical-mediated lipid peroxidation, on muscle samples taken at the conclusion of fatigue trials. As a control, we also measured TBAR concentrations for muscle samples taken from nonfatigued diaphragm. We found that the rate of development of diaphragm fatigue was much greater in saline and denatured PEG-SOD-treated groups than for animals pretreated with either PEG-SOD or DMSO, with force falling to 23 +/- 4, 21 +/- 4, 50 +/- 7, and 47 +/- 6% of its initial value, respectively, over a 2-h period of electrophrenic stimulation in these four groups of animals (p < 0.01). TBAR concentrations in fatigued diaphragm from saline and denatured PEG-SOD-treated animals were significantly higher than levels for either nonfatigued fresh diaphragm or fatigued diaphragm taken from PEG-SOD- or DMSO-treated animals (p < 0.01). These data suggest that diaphragm fatigue resulting from repetitive low-frequency stimulation is associated with lipid peroxidation within this muscle and that pretreatment with free radical scavengers prevents lipid peroxidation and reduces the rate of development of fatigue.     

Conformation induction in melanotropic peptides by trifluoroethanol: fluorescence and circular dichroism study

Studies on the anticonvulsant efficacy of the major antiepileptic drug phenytoin in kindled rats have often reported inconsistent effects. It has been proposed that technical and genetic factors or poor and variable absorption of phenytoin after i.p. or oral administration may be involved in the lack of consistent anticonvulsant activity of phenytoin in this model of temporal lobe epilepsy. We examined if kindling itself changes the anticonvulsant efficacy of phenytoin by testing this drug before and after amygdala kindling in male and female Sprague-Dawley rats. To exclude the possible bias of poor and variable absorption, blood was sampled in all experiments for drug analysis in plasma. The threshold for induction of focal seizures (afterdischarge threshold; ADT) was used for determining phenytoin's anticonvulsant activity. Before kindling, phenytoin, 75 mg/kg i.p., markedly increased ADT in both genders, although the effect was more pronounced in males. Following kindling, the anticonvulsant activity obtained with phenytoin, 75 mg/kg, before kindling was totally lost, and female rats even exhibited a proconvulsant effect upon administration of this dose, indicating that kindling had dramatically altered the anticonvulsant efficacy of phenytoin. Plasma levels of phenytoin were comparable before and after kindling, and were within or near to the 'therapeutic range' known from epileptic patients. When the dose of phenytoin was reduced to 50 or 25 mg/kg i.p., significant anticonvulsant effects on ADT were obtained. When phenytoin, 50 mg/kg, was administered i.p. or i.v. in the same group of fully kindled rats, both anticonvulsant activity and plasma drug levels were comparable with both routes, indicating that the i.p. route is suited for such studies. The data indicate that kindling alters the dose-response of phenytoin in that a high anticonvulsant dose becomes ineffective or proconvulsant after kindling, possibly by an increased sensitivity of the kindled brain to proconvulsant effects of phenytoin which normally only occur at much higher doses. If similar alterations evolve in humans during development of chronic epilepsy, this may be involved in the mechanisms leading to intractability of temporal lobe epilepsy.     

Teratogenicity of 13-cis retinoic acid and phenobarbital sodium in CF-1 mice

The teratogenicity of 13-cis-retinoic acid (RA) either administered alone or following pretreatment with phenobarbital sodium (PB), was assessed. Groups of gravid CF-1 mice were administered dosages of either 10, 100, 200, or 400 mg/kg of RA orally on either days 11, 12 or 13 of gestation, in order to determine structural alterations. In addition, separate groups of mice were orally pretreated with 80 mg/kg/day of PB on days 7 through 10 of gestation prior to the administration of RA. Skeletal alterations attributed to maternal administration of either 100, 200 or 400 mg/kg of RA on days 11-13 included delayed ossification of the limbs and supraoccipital bone, the presence of extra ribs, and various sternebral defects. Soft tissue alterations included cleft palate and dilation of the renal pelves which occurred following maternal exposure on days 11 and 12-13, respectively. Significant decreases in the incidence of cleft palate and delayed ossification of the limbs were observed in those dams administered RA on days 11 or 12, respectively, following prior treatment with PB. These data suggest that administration of PB, a prototypical hepatic microsomal enzyme inducer, may partially antagonize the teratogenicity of RA.     

In situ detection of myocardial infarction in pig by measurements of aspartate aminotransferase (ASAT) activity in the interstitial fluid

Recent studies have suggested that the fetal dysmorphogenesis in diabetic pregnancies is associated with an increase in embryonic oxygen-free radicals. This excess of oxygen-free radicals may result from either overproduction or decreased clearance by the enzymatic scavenging mechanism. However, there are no in vivo data on the activity of embryonic oxygen-free radical scavenging enzymes. The purpose of the current study is to investigate whether this increase in embryonic oxygen-free radicals is the result of a change in the activity of the fetal oxygen-free radical scavenging/antioxidant enzymes during pregnancy complicated by maternal diabetes in an in vivo rat model. Thirty-six Sprague-Dawley rats were randomly assigned to one of two study groups: nondiabetic control and an untreated diabetic group. On day 12, fetuses were examined for crown-rump lengths, somite numbers, and external anomalies. The activity of fetal oxygen-free radical scavenging enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), were determined. The untreated diabetic group of rats had a significantly higher mean blood glucose level than that of the nondiabetic controls and also a significantly lower weight gain, higher resorption rate, smaller embryonic size with lower total protein content, and a approximately 6-fold increase in the rate of fetal neural tube defects compared to the nondiabetic controls. Superoxide dismutase activity was significantly reduced in the embryos with neural tube defects regardless of maternal diabetic status (2.25 +/- 0.83 vs. 1.17 +/- 0.04 u/mg protein; P < 0.05). Glutathione peroxidase and catalase activity were significantly reduced in malformed versus normal-formed embryos of nondiabetic mothers (GPX-2.68 +/- 1.15 vs. 4.46 +/- 1.12 mu/mg protein, CAT -1.67 +/- 0.53 vs 2.49 +/- 0.61 u/mg protein respectively; P < 0.01). However, overall catalase activity was increased in embryos of diabetic mothers as compared to controls. Two-way analysis of variance identified fetal malformations as the variance associated with reduced fetal SOD activity, whereas maternal diabetes was associated with the increase in fetal catalase activity. Neither neural tube defect nor maternal diabetes was found to be the variable affecting fetal GPX activity, Fetal oxygen-free radical scavenging enzymes respond differently to the adverse environment created by maternal diabetes during pregnancy. Defects in embryonic SOD and catalase activity, regardless of maternal diabetic status, may reduce the ability of the fetus to clear free oxygen radicals, thereby exposing it to an increased oxidative load that may cause fetal dysmorphogenesis. The diabetic state of the mothers did not decrease embryonic activity of any of the scavenging enzymes. Therefore, although excess oxidative load, as observed in diabetes, may cause tissue injury and embryopathy, the mechanism does not appear to be a diabetes-induced reduction in the action of the scavenging enzymes.     

The effects of lithium on neurulation stage mouse embryos

The aim of this study was to evaluate the maternal toxicity and teratogenicity of lithium following intraperitoneal injection (i.p.) with lithium carbonate (Li2CO3) in pregnant CD-1 mice at the developmental stage of neurulation (E8; day of vaginal plug, E0). Light (LM) and electron (TEM) microscopic studies were also done to document the tissue and cellular changes occurring in embryonic tissues during the 48 h following treatment with 300 mg/kg body wt. Li2CO3. Controls were untreated or given equimolar amounts of NaCl or Na2CO3. A pharmacokinetic study showed that lithium was rapidly absorbed from the peritoneal cavity after the above-stated dose, achieved peak serum levels of 9.8 mmol/l within 1 h, had a half-life in the blood of 5 h and was completely cleared by 16 to 24 h after injection. Doses of Li2CO3 > 300 mg/kg body wt. were toxic to adult CD-1 mice. The latter dose had no detectable maternal toxicity but caused a 19% resorption rate and 2% incidence of open cranial neural tube defect in gestations terminated on E18. The malformation and resorption rates in gestations terminated on E11, E12 and E14 were not significantly different from those of E18. A strong litter effect was seen both for the resorption and malformation rates at all stages examined. At 3 h after treatment cell death became evident in the neuroepithelium. Cells continued to die for approximately 17 h and all necrotic debris had been cleared by 48 h. Also at 3 h after treatment small densely stained inclusions began to appear in mesodermal cells. TEM showed these to be non-membrane bound with an irregular shape and variable size; the lack of staining for acid phosphatase indicated a non-lysosomal structure; the ultrastructural features suggested a lipoid basis. At 24 h after treatment vascular ruptures and surface ectodermal ruptures were seen in the cranial mesoderm. These ruptures with extravascated blood were also seen at 48 h after treatment. A litter effect was also noted with respect to the tissue and cellular changes. These experiments suggest that the developing vascular system may be a target for lithium. In addition, the possibility is discussed that lithium induced cell death in the neuroepithelium may lead to neural tube defects.     

Clinical and antiinflammatory effects of intranasal budesonide aqueous pump spray in the treatment of perennial allergic rhinitis

BACKGROUND: Neutrophils are of great importance for the host's defense against invading organisms. Granulocyte colony-stimulating factor (G-CSF) has been used to augment both the neutrophil number and function, and its prophylactic administration has proved beneficial in animal models of sepsis. However, pretreatment with G-CSF is not practical under clinical conditions. We therefore investigated the effect of recombinant human (rh)G-CSF, administered only after infection, on the survival rate as well as the hemodynamic and cytokine response of the animals. METHODS: Chronically catheterized conscious pigs were challenged with Pseudomonas aeruginosa (8 x 10(7) colony-forming units kg(-1) x h(-1) for 120 h (control group, n = 10). Animals in the G-CSF group (n = 7) also received rhG-CSF (5 microg kg(-1) x day(-1)), the first dose being given 3 h after beginning bacterial infusion. RESULTS: The mortality rate was 50% (5/10) and 29% (2/7) in the control and G-CSF groups, respectively (p = NS, control vs. G-CSF group). Fever, severe pulmonary hypertension, and a hyperdynamic response were recorded in all of the animals. In spite of a prompt and significant recovery from the initial leukopenia (p < .05 vs. control group), the animals of the G-CSF group showed no significant differences in the parameters investigated from those of the controls. Compared with the survivors, the interleukin-1 receptor antagonist was markedly elevated in all nonsurvivors after 6 h of sepsis (p < .05). CONCLUSIONS: These data suggest that treatment with rhG-CSF after the onset of bacterial sepsis might not significantly improve the chances of survival for non-neutropenic patients.     

Maternal hyperoxia greatly reduces the incidence of phenytoin-induced cleft lip and palate in A/J mice

The A/J mouse has been used to study the teratogenic affects of phenytoin. The developmental abnormalities produced in offspring of this model are similar to some of the malformations observed in cases of human "fetal hydantoin syndrome." Placing pregnant A/J mice in a hyperoxic chamber after phenytoin injection greatly reduces the incidence of phenytoin-induced cleft lip and palate. These results suggest that phenytoin may affect embryonic development indirectly by altering maternal physiology. This maternally mediated mechanism, and the protection against it afforded by hyperoxia, has general implications for the effects of maternal toxicity on teratogenesis.     

Rat Hsp20 confers thermoresistance in a clonal survival assay, but fails to protect coexpressed luciferase in Chinese hamster ovary cells

The effect of stress-like concentrations of cortisol (C) on estrogen-dependent expression of GnRH receptor was evaluated using orchidectomized sheep (wethers; n = 6 animals per group). C (5.0 mg/50 kg per hour; groups 1-4) or a comparable volume of vehicle (groups 5-8) was delivered by continuous infusion for 48 h. During the final 24 h of infusion, animals received concurrent infusion of estradiol (E2) at rates of 0 (groups 1 and 5), 0.5 (groups 2 and 6), 2.0 (groups 3 and 7), or 8.0 (groups 4 and 8) microg/50 kg per hour. Pituitary tissue was collected at the end of infusion. Although C did not affect (p > 0.05) the basal concentration of GnRH receptor or GnRH receptor mRNA, it reduced (p < 0.05) the increase in receptor and receptor mRNA induced by concurrent administration of 0. 5 microg E2/50 kg per hour. In contrast, the increase in GnRH receptor expression induced by higher levels of estrogen stimulation was not affected (p > 0.05) by concurrent administration of C. The effect of C on the temporal pattern of E2-dependent increase in GnRH receptor expression was assessed using wethers receiving E2 (0.5 microg/50 kg per hour) by continuous infusion for 0 (groups 1 and 5), 24 (groups 2 and 6), 48 (groups 3 and 7), or 72 h (groups 4 and 8). Animals received C (5.0 mg/50 kg per hour; groups 1-4) or vehicle (groups 5-8) beginning 24 h before, and continuing throughout, the E2 delivery period. Stress-like concentrations of C reduced (p < 0. 05) the increase in GnRH receptor and receptor mRNA induced after 24 h of E2 stimulation. However, the suppressive effect of C was transient, and tissue levels of GnRH receptor and receptor mRNA were comparable after 72 h of E2 infusion in animals receiving C or vehicle alone. Collectively these observations demonstrate that C suppresses estrogen-dependent increase in tissue concentrations of GnRH receptor and receptor mRNA. However, this effect of C is transient and not evident in animals receiving moderate to high levels of estrogen stimulation. This transient suppression of GnRH receptor expression may account, at least in part, for the anti-gonadal effect of glucocorticoids.     

Magnesium sulfate versus phenytoin for seizure prevention in amygdala-kindled rats

OBJECTIVE: Magnesium sulfate is widely used for seizure prophylaxis in preeclampsia-eclampsia. However, its anticonvulsant effects in other types of seizures have not been proved. Diphenylhydantoin has been widely characterized as the "gold standard" of anticonvulsants. In this study we compared the anticonvulsant effects of therapeutic blood levels of magnesium sulfate and phenytoin in seizures generated in amygdala-kindled rats. STUDY DESIGN: Eighteen male rats had a bipolar electrode stereotaxically implanted into the central nucleus of the amygdala. After recovery an electrical seizure threshold was determined for each rat. Rats were stimulated twice daily at their seizure thresholds (i.e., kindling) until three consecutive generalized tonic-clonic seizures occurred. Kindled rats randomly received one of the following intravenous injections in a volume of 1.5 ml/kg: saline solution, magnesium sulfate (30, 60, or 90 mg/kg), or phenytoin (12.5, 25, or 50 mg/kg). Fifteen minutes after injection rats were stimulated at their seizure thresholds, and electrical and behavioral seizure activity was assessed. Statistical comparisons were made by analysis of variance and post hoc comparisons when appropriate. RESULTS: Magnesium sulfate had no effect on any of the seizure parameters assessed. Phenytoin significantly reduced seizure duration (p < 0.01), duration of postictal depression (p < 0.01), and behavioral seizure stage (p < 0.01). CONCLUSION: Amygdala-kindled seizures are more potently inhibited by phenytoin than by magnesium sulfate.     

Molecular-weight-dependent effects of nonanticoagulant heparins on allergic airway responses

We have hypothesized that antiallergic activity of inhaled heparin is molecular weight dependent and mediated by "nonanticoagulant fractions" (NAF-heparin). Therefore, we studied comparative effects of high-, medium-, and ultralow-molecular-weight (HMW, MMW, and ULMW, respectively) NAF-heparins on acute bronchoconstrictor response (ABR) and airway hyperresponsiveness (AHR) in allergic sheep. Specific lung resistance was measured in 23 allergic sheep, before and immediately after challenge with Ascaris suum antigen, without and after pretreatment with inhaled NAF-heparins. Airway responsiveness was estimated before and 2 h postantigen as the cumulative provocating dose of carbachol in breath units, which increased specific lung resistance by 400%. NAF-heparins attenuated ABR and AHR in a molecular-weight-dependent fashion. HMW NAF-heparin (n = 8) was the least effective agent: it attenuated ABR [inhibitory dose causing 50% protection (ID50) = 4 mg/kg] but had no effect on AHR. MMW NAF-heparin (n = 8) showed intermediate efficacy (ABR ID50 = 0.8 mg/kg, AHR ID50 = 1.4 mg/kg), whereas ULMW NAF-heparin (n = 7) was the most effective agent (ABR ID50 = 0.4 mg/kg, AHR ID50 = 0.2 mg/kg). ULMW NAF-heparin was 3.5 times more potent in attenuating antigen-induced AHR when administered "after" antigen challenge and failed to inhibit the bronchoconstrictor response to carbachol and histamine. In 15 additional sheep, segmental antigen challenge caused a marked increase in histamine in bronchoalveolar lavage fluid that was not prevented by any of the inhaled NAF-heparins. These data indicate that antiallergic activity of inhaled heparin is independent of its anticoagulant action and resides in the <2,500 ULMW chains. The antiallergic activity of NAF-heparins is mediated by an unknown biological action and may have therapeutic potential.     

In vivo characterization of T-794, a novel reversible inhibitor of monoamine oxidase-A, as an antidepressant with a wide safety margin

T-794 is a new reversible inhibitor of MAO type A. In order to predict its clinical utility as an antidepressant, we examined its pharmacological profile (i.e., MAO inhibitory activity, antidepressant-like activity and safety) in vivo in rodents. The p.o. administration of T-794 potentiated L-5-hydroxytryptophan-induced symptoms with ED50 = 1.01 mg/kg (mice) or 1.15 mg/kg (rats), and L-dopa-induced behavior with ED50 = 5.90 mg/kg (mice), whereas it did not alter the effect of beta-phenylethylamine even at 100 mg/kg (mice). In the L-5-hydroxytryptophan test in rats, the activity of T-794 (at twice the dose of ED50) disappeared by 8 h; the duration of action was similar to that of moclobemide. These results confirm the previous biochemical results that MAO-A inhibition by T-794 is highly selective and of short duration. T-794 was effective in three animal models of depression: reserpine reversal (mice, rats), behavioral despair test (mice) and learned helplessness (rats). In these tests, it had potency similar to or greater than moclobemide, tranylcypromine or imipramine. The p.o. administration of T-794 (30 mg/kg) did not affect the pressor effect of tyramine in anesthetized rats, whereas moclobemide (30 mg/kg) and tranylcypromine (6 mg/kg) potentiated the effect. Acute toxicity of T-794 proved to be very low (maximal tolerated dose > 2 g/kg p.o.) in contrast to brofaromine (maximal tolerated dose = 150 mg/kg p.o.). Unlike tricyclic antidepressants, T-794 did not prevent the oxotremorine-induced tremor even at 100 mg/kg p.o.; in this it demonstrated a lack of the anticholinergic activity. These results suggest that T-794 is an effective and particularly safe antidepressant and that it may make an important contribution in the treatment of depressive disorders.     

Effect of selenium-enriched yeast pretreatment on the antioxidative defense in the skin of rats exposed to heat shock

Skin protection against heat shock and the specificity in the organization of antioxidative defenses were examined in rats given oral antioxidative pretreatment with selenium (Se)-enriched yeast and vitamins E, C, and A for 15 days and then exposed to hyperthermia. The activity of antioxidative enzymes in the skin and the liver was monitored 1 hour and 3 hours after heat shock. Glutathione peroxidase (GSH-Px) activity was increased in the skin after heat shock in the groups supplemented with antioxidants, but not in the controls. In contrast, the activity of liver GSH-Px was increased only in the controls receiving antioxidants. Heat shock led to a decrease in liver superoxide dismutase (SOD) activity at 1 hour in the antioxidant-supplemented group, but this was unchanged in the liver of all other groups and in the skin. The activity of thioredoxin reductase (TR) in the skin was increased in the antioxidant supplemented group 1 hour after heat shock, whereas the hepatic thioredoxin reductase activity was decreased. The activities of catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) were unaffected by either treatment. These results suggest that supplementation with antioxidants protects the skin against heat shock, especially with respect to the GSH-Px and TR activity. The different response of the skin in comparison with the liver probably reflects differences in organization and regulation of antioxidative defenses.     

Peripheral administration of cholecystokinin activates c-fos expression in the locus coeruleus/subcoeruleus nucleus, dorsal vagal complex and paraventricular nucleus via capsaicin-sensitive vagal afferents and CCK-A receptors in the rat

Intraperitoneal (i.p.) administration of sulfated CCK octapeptide (CCK-8S) has been shown to induce changes in neuronal activity in the nucleus of the solitary tract (NTS) and area postrema (AP), sensory parts of the dorsal vagal complex (DVC), and in the paraventricular nucleus of the hypothalamus (PVN), as determined by activation of c-fos expression. Whether peripheral CCK influences neuronal activity in the locus coeruleus (LC)/subcoeruleus nucleus (SC) was investigated in awake rats at intraperitoneal (i.p.) injection of CCK-8S by c-Fos immunohistochemistry. CCK-8S i.p. (25, 50, and 100 micrograms/kg, respectively) dose-dependently increased the average number of c-Fos-LI-positive cells/section in the LC/SC by the factor 5.9, 8.2, and 11.7, respectively. Pretreatment with the CCK-A receptor antagonist MK-329 (devazepide; 1 mg/kg and 2 mg/kg i.p.) reduced the CCK-induced increase in c-fos expression in the LC/SC by 54% and 75%, respectively; the CCK-B receptor antagonist L-365,260 had no effect. Perivagal capsaicin pretreatment diminished the CCK-induced increase in the number of c-Fos-LI-positive cells in the LC/SC by 65%. In comparison, the CCK-A antagonist devazepide (1 mg/kg and 2 mg/kg i.p.) reduced the increase in c-fos expression by 76% and 88% in the PVN, 69% and 88% in the NTS, 86% and 83%, respectively, in the AP. Capsaicin diminished the CCK-induced increase in c-Fos-LI-positive cells in the PVN by 64%, in the NTS by 60%, but in the AP only by 25%. Immunostaining against the nuclear antigen c-Fos and the cytoplasmatic antigen tyrosine hydroxylase (TH) showed that 40% of all c-Fos-LI-positive cells in the LC/SC were TH-LI positive at 25 micrograms CCK/kg. The data indicate that CCK-8S i.p. induces modulation of neuronal activity in the LC/SC, DVC and PVN predominantly by peripheral action of CCK-A receptors and capsaicin-sensitive vagal afferents. These findings suggest that the LC/SC is involved in CNS-mediated regulatory influences of peripheral CCK.     

Influence of pentoxifylline on fevers induced by bacterial lipopolysaccharide and tumor necrosis factor-alpha in guinea pigs

In guinea pigs intraperitoneal (i.p.) injections of 50 mg/kg pentoxifylline had no influence on abdominal temperature while higher doses of pentoxifylline caused a hypothermic response lasting for 2-3 h. Administration of 50 mg/kg pentoxifylline 1 h before intramuscular (i.m.) injections of 20 micrograms/kg bacterial lipopolysaccharide reduced the lipopolysaccharide-induced production of endogenous tumor necrosis factor-alpha (TNF-alpha) by 68%. The second phase of lipopolysaccharide-induced fever was significantly attenuated by pretreatment with 50 mg/kg pentoxifylline, a dose which had, per se, no influence on core temperature of guinea pigs. The thermal response of guinea pigs to administration of exogenous TNF-alpha was not modulated by pretreatment with pentoxifylline. Intra-arterial infusions with 5 micrograms/kg TNF-alpha, a dose which yielded the same circulating TNF bioactivity as i.m. injections of 20 micrograms/kg lipopolysaccharide, induced a biphasic febrile response. The magnitude and duration of TNF-induced fever were the same whether guinea pigs were pretreated with pentoxifylline or with 0.9% saline. The results indicate that endogenous formation of TNF-alpha may contribute to the development of fever induced by lipopolysaccharide, but is not its only mediator, since the first phase of lipopolysaccharide-induced fever was not altered by the blockade of TNF production.     

Design, synthesis, and anticonvulsant activity of 1-(pyrid-3-ylsulfonamido)-2-nitroethylenes

The lipophilic 1-cycloalkylamino-1-(pyrid-3-yl-sulfonamido)-2-nitr oethylenes were synthesized as bioisosteres of BM-34, an anticonvulsant sulfonylthiourea. Compound 17 (ip) emerged from the maximal electroshock seizure (MES) test with a 50% effective dose (ED50) of 8.25 mg/kg. Its anticonvulsant profile was similar to that of phenytoin (ED50 = 9.51 mg/kg) and of BM-34 (ED50 = 1.19 mg/kg): active in the MES test and inactive in seizures induced by subcutaneous injection of pentetrazole, strychnine, bicuculline, picrotoxin, or N-methyl-D,L-aspartate. The neurotoxicity of 17 (TD50 = 113.8 mg/kg) was lower than that of phenytoin (TD50 = 65.5 mg/kg) but higher than that of BM-34 (TD50 = 147.2 mg/kg). Crystallographic study revealed that BM-401 (17) was a zwitterionic structure. Its sulfonamido nitroethylene side chain adopted a conformation which placed the two cycloalkyl rings face to face to form a single hydrophobic area.     

Abnormal functional connectivity in Alzheimer''s disease: intrahemispheric EEG coherence during rest and photic stimulation

Tolbutamide is a sulfonylurea oral hypoglycaemic agent with suspected teratogenicity in humans and demonstrated teratogenicity in laboratory animals, but the underlying mechanism is unknown. This study examined maternal-to-conceptus tolbutamide transfer on gestational days 9.5 and 10.5 and drug concentration in embryonic head, heart, and trunk regions on gestational day 10.5 after maternal dosing in mouse. Embryos exposed to tolbutamide in vitro on gestational day 8.5 were assayed for glucose uptake, glycolysis, and protein content after 6, 12, and 24 hr. Dose-dependent tolbutamide transfer from maternal serum to extraembryonic fluid occurred on gestational day 9.5 and 10.5, with highest tolbutamide levels in embryonic heart on gestational day 10.5. In vitro tolbutamide exposure on gestational day 8.5 decreased glycolysis at 6 hr, increased glycolysis at 24 hr, and had no effect on glucose uptake at 6, 12, or 24 hr. Embryonic protein content reflected growth retardation after 24 hr tolbutamide exposure. Thus, mouse embryos are directly exposed to tolbutamide after maternal dosing on gestational day 9.5 and 10.5, with concentration of drug within embryonic heart. Tolbutamide-induced changes in glucose metabolism are less apparent in whole embryos than reported in adult tissues.