Tultul Nayyar

Alteration of pregnancy related hormones and fetal survival in F-344 rats exposed by inhalation to benzo(a)pyrene

The objective of this study was to evaluate the effect of subacute exposure to inhaled benzo(a)pyrene (BaP) on fetal survival and luteal maintenance using timed-pregnant Fisher 344 rats. Prior to assignment of pregnant rats to treatment and control groups, numbers of implantation sites were determined on gestation day (GD) 8 via midventral laparotomy. Subsequently, animals were assigned randomly to three treatment groups and two control groups. Treatment consisted of subacute exposure of rats via inhalation to BaP 25, 75, and 100 micro g/m(3), 4h daily for 10 days (GD-11-20). Control animals were either sham exposed to carbon black (CB) to control for inert BaP carrier or remained unexposed (UNC). Blood samples were collected on days 15 and 17 of gestation via sinus orbital veini-puncture for plasma. Number of pups per litter was determined postpartum and fetal survival rate was expressed as a percentage of the corresponding implantation sites. Radioimmunoassays were used to determine plasma progesterone, estrogen, and prolactin (indirect measurement of decidual luteotropin) concentrations. Fetal survival among BaP-treated rats declined in a dose-dependent manner (25 micro g/m(3), 78.3% per litter; 75 micro g/m(3), 38.0% per litter; 100 micro g/m(3), 33.8% per litter; P<0.05) compared with CB (96.7% per litter) and UNC (98.9% per litter). Plasma progesterone, estrogen, and prolactin concentrations also declined as a result of subacute exposure of rats to BaP compared to controls. These data suggest that inhaled BaP compromised fetal survival and consequently luteotropic activity in the exposed animals.

MODULATION IN THE DEVELOPMENTAL EXPRESSION PROFILE OF Sp1 SUBSEQUENT TO TRANSPLACENTAL EXPOSURE OF FETAL RATS TO DESORBED BENZO[a]PYRENE FOLLOWING MATERNAL INHALATION

Any alteration of the critical sequence of genes that are required to coordinate the differentiation of cells, the promotion of migration, dendritic arborization, synapse formation, and myelination in the developing nervous system would be expected to have deleterious consequences. The focus of this article is a molecular evaluation of the neurotoxicological effects that result subsequent to the transplacental exposure of fetal rats to desorbed benzo(a)pyrene (BaP) following maternal inhalation. A state-of-the-art, newly designed, fabricated, and tested model aerosol generation system was utilized in these studies. Timed-pregnant Sprague Dawley rats were exposed for 4 h on gestation day 15 of a 21-day gestation period to an acute dose of BaP: carbon black aerosol (100 µg/m3). Controls received carbon black only. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The aerosol exhibited a trimodal distribution with 95% cumulative mass less than 15.85 µm, 90% cumulative mass less than 10 µm, 67.5% cumulative mass less than 2.5 µm and 66.2% cumulative mass less than 1.0 µm. Time-course bioavailability results indicated that greater than 95% of the parent compound is cleared from blood 240 min postexposure. An Sp1 transcription factor consensus sequence was examined by electrophoretic mobility shift analysis of nuclear extracts from various brain regions of resulting pups on postnatal days 3, 5, 7, 10, and 15. It revealed perturbations in the developmental expression profile of Sp1 abundance as a result of nose-only particulate aerosol exposure to the timed-pregnant dam. The data obtained on the temporal and spatial regulation of gene expression in the brain indicate that (1) Sp1 DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a consequence of the transplacental deposition of desorbed BaP to the fetus is in utero neurotoxicity.Any alteration of the critical sequence of genes that are required to coordinate the differentiation of cells, the promotion of migration, dendritic arborization, synapse formation, and myelination in the developing nervous system would be expected to have deleterious consequences. The focus of this article is a molecular evaluation of the neurotoxicological effects that result subsequent to the transplacental exposure of fetal rats to desorbed benzo(a)pyrene (BaP) following maternal inhalation. A state-of-the-art, newly designed, fabricated, and tested model aerosol generation system was utilized in these studies. Timed-pregnant Sprague Dawley rats were exposed for 4 h on gestation day 15 of a 21-day gestation period to an acute dose of BaP:carbon black aerosol (100 microg/m(3)). Controls received carbon black only. Nominal and chamber concentrations of the particulate aerosol were determined gravimetrically with a seven-stage cascade impactor. The aerosol exhibited a trimodal distribution with 95% cumulative mass less than 15.85 microm, 90% cumulative mass less than 10 microm, 67. 5% cumulative mass less than 2.5 microm and 66.2% cumulative mass less than 1.0 microm. Time-course bioavailability results indicated that greater than 95% of the parent compound is cleared from blood 240 min postexposure. An Sp1 transcription factor consensus sequence was examined by electrophoretic mobility shift analysis of nuclear extracts from various brain regions of resulting pups on postnatal days 3, 5, 7, 10, and 15. It revealed perturbations in the developmental expression profile of Sp1 abundance as a result of nose-only particulate aerosol exposure to the timed-pregnant dam. The data obtained on the temporal and spatial regulation of gene expression in the brain indicate that (1) Sp1 DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a consequence of the transplacental deposition of desorbed BaP to the fetus is in utero neurotoxicity.

Assessment of metabolites and AhR and CYP1A1 mRNA expression subsequent to prenatal exposure to inhaled benzo(a)pyrene

Few studies have focused on environmental aerosol contaminant, mechanistically‐based, dose‐related neurotoxicity with respect to development of the central nervous system. To fill this important data gap and to highlight possible mechanistic pathways, a study was undertaken to determine metabolite concentrations associated with the transplacental disposition of inhaled benzo(a)pyrene (B(a)P) and the resulting effects on the status of aryl hydrocarbon receptor (AhR), and cytochrome P450 1A1 (CYP1A1) mRNA in preweaning F1 generation animals. In this study, laparotomy on GD 8 was performed on timed‐pregnant rats followed by dosing via nose‐only exposure for 4 h a day for 10 days (GD 11–GD 20) to three concentrations of a B(a)P: carbon black aerosol (25, 75 and 100 μg/m3). A dose‐dependent decrease in birth index was observed in the B(a)P exposed group as compared to the controls (P<0.05). Analysis of cerebrocortical extracts from F1 generation pups revealed a dose‐dependent (P<0.05) increase in total B(a)P metabolites. Analysis of cerebrocortical and hippocampal mRNA developmental expression profiles for AhR and CYP1A1 using 18sRNA as the internal standard, revealed that inhaled B(a)P upregulates AhR during the first postnatal month. The present study suggest that prenatal exposure to inhaled B(a)P upregulates hepatic aryl hydrocarbon receptor dependent mechanisms in the F1 generation. Hepatic upregulation of the aryl hydrocarbon receptor may modulate the potential for benzo(a)pyrene toxicity via the activation of cytochrome P450 and the subsequent deposition of lipophillic metabolites to developing central nervous system structures such as cerebral cortex and hippocampus.

Cortical serotonin and norepinephrine denervation in parkinsonism: Preferential loss of the beaded serotonin innervation

Parkinson’s Disease (PD) is marked by prominent motor symptoms that reflect striatal dopamine insufficiency. However, non‐motor symptoms, including depression, are common in PD. It has been suggested that these changes reflect pathological involvement of non‐dopaminergic systems. We examined regional changes in serotonin (5‐HT) and norepinephrine (NE) systems in mice treated with two different 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treatment paradigms, at survival times of 3 or 16 weeks after the last MPTP injection. MPTP caused a decrease in striatal dopamine concentration, the magnitude of which depended on the treatment regimen and survival interval after MPTP treatment. There was significant involvement of other subcortical areas receiving a dopamine innervation, but no consistent changes in 5‐HT or NE levels in subcortical sites. In contrast, we observed an enduring decrease in 5‐HT and NE concentrations in both the somatosensory cortex and medial prefrontal cortex (PFC). Immunohistochemical studies also revealed a decrease in the density of PFC NE and 5‐HT axons. The decrease in the cortical serotonergic innervation preferentially involved the thick beaded but not smooth fine 5‐HT axons. Similar changes in the 5‐HT innervation of post‐mortem samples of the PFC from idiopathic PD cases were seen. Our findings point to a major loss of the 5‐HT and NE innervations of the cortex in MPTP‐induced parkinsonism, and suggest that loss of the beaded cortical 5‐HT innervation is associated with a predisposition to the development of depression in PD.

Transplacental effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the temporal modulation of Sp1 DNA binding in the developing cerebral cortex and cerebellum.

Gestational exposure to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, (dioxin) often leads to neurodevelopmental and neurobehavioral deficits, which clearly suggest the involvement of the central nervous system. The objective of this study was to determine the effects of an acute, gestational exposure to dioxin on the developmental expression profile of a transcription factor (Sp1) that is involved in growth and differentiation in the developing brain of F, generation pups. Timed-pregnant Harlan Sprague Dawley rats were exposed to single oral doses of 0.25, 0.5, and 1.0 microg dioxin/kg body weight or vehicle corn oil on gestation day 15. Electrophoretic mobility shift assays (EMSA) of the nuclear extracts from the cerebral cortex and cerebellum of the resulting pups, on postnatal days (PND) 3.5, 10, 15, 20 and 30, with an Sp1 consensus oligonucleotide sequence revealed modulations in the DNA binding activity of Sp1 as a result of the transplacental dioxin exposure. In the cerebral cortex, a dose-dependent premature peak of Sp1 DNA-binding on PND 3 was observed when compared with the control animals where Sp1 DNA-binding was maximal on PND 10. In the postnatally-developing cerebellum, a dose-dependent premature peak of Sp1 DNA binding on PND 5 was observed when compared with the control animals where Spl DNA-binding was maximal on PND 15. The mean birth index, body weight gain, and gross brain weight of the pups were not statistically different from the control animals over the 30 day postnatal period studied. The data obtained on the Sp1 developmental expression profiles in the brain indicate that (1) Sp I DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a potential consequence of the transplacental effect of dioxin to the fetus is in utero neurotoxicity.

Identification of calcium-dependent phospholipid-binding proteins (annexins) from guinea pig alveolar type II cells

A new group of calcium-regulating proteins, called annexins or Ca++-dependent phospholipid-binding proteins (PLBP), have been detected in different species, organs and cell types. In the present study, we have identified and quantitated PLBP from guinea pig lung, lavage fluid and alveolar type II cells to elucidate the possible role of PLBP in lung surfactant biogenesis and secretion. Lungs were lavaged and type II cells from lavaged lung were isolated by elastase digestion and purified by centrifugal elutriation. For the quantitative identification of PLBP, we performed ELISA assays and Western blot analysis by using an antiserum raised in guinea pigs against a pure rabbit lung 36 kDa PLBP. The lavage fluid, cytosol from lung and type II cells contained 784,167 and 435 ng per mg protein, respectively, of PLBP. The SDS-PAGE electrophoretic pattern and Western blot confirmed that all lung samples have band corresponding to a 36 kDa protein. This indicates that both alveolar type II cells and lavage fluid have higher levels of PLBP than whole lung cytosol.

MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE EXPOSURE INCREASES RETINOL IN GUINEA PIG LUNGS

We have studied in guinea pigs the effects of cigarette smoke exposure on vitamin A (retinol) levels in plasma, lung, lung lavage, and liver. Smoke was generated from 1R3F cigarettes in a smoke exposure instrument designed by University of Kentucky Tobacco and Health Research Institute. Three-week-old male guinea pigs were exposed to mainstream, sidestream, or sham smoke, generated twice daily from three cigarettes for 6 weeks. In addition, some animals were kept as room controls for some time. After 6 weeks of smoke exposure, some animals were allowed to recover for 6 weeks without smoke. After 6 weeks of smoking, the plasma retinol levels were lower in both smoke exposed groups when compared to the values in the sham group. Furthermore, in comparison to the sham group, the mainstream and sidestream smoke exposed groups showed a 7.6- and 8.3-fold increase in the levels of lung retinol, respectively. After the 6-week recovery period, plasma retinol of both smoke-exposed groups reached the control levels. In contrast, withdrawal of smoking did not show such an effect on the lung retinol level in both mainstream or sidestream groups. Electronmicroscopy of the lungs showed deleterious alterations in the morphology of the lungs in both mainstream and sidestream groups. Although the mechanism(s) involved in the elevation of retinol content of the lung due to smoke exposure remains to be elucidated, it is of interest that elevation of retinol content and alteration of lung morphology occurred not only in the mainstream smoke exposed but also in the sidestream group.

The 5-HT2A Receptor Antagonist M100907 Produces Antiparkinsonian Effects and Decreases Striatal Glutamate

5-HT plays a regulatory role in voluntary movements of the basal ganglia and has a major impact on disorders of the basal ganglia such as Parkinsons disease (PD). Clinical studies have suggested that 5-HT2 receptor antagonists may be useful in the treatment of the motor symptoms of PD. We hypothesized that 5-HT2A receptor antagonists may restore motor function by regulating glutamatergic activity in the striatum. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibited decreased performance on the beam-walking apparatus. Peripheral administration of the 5-HT2A receptor antagonist M100907 improved performance of MPTP-treated mice on the beam-walking apparatus. In vivo microdialysis revealed an increase in striatal extracellular glutamate in MPTP-treated mice and local perfusion of M100907 into the dorsal striatum significantly decreased extracellular glutamate levels in saline and MPTP-treated mice. Our studies suggest that blockade of 5-HT2A receptors may represent a novel therapeutic target for the motor symptoms of PD.

Reverse microdialysis of a 5-HT2A receptor antagonist alters extracellular glutamate levels in the striatum of the MPTP mouse model of Parkinson’s disease

Clinical observations have suggested that antagonism of 5-HT2A receptors may benefit patients with parkinsonian symptomatology. The mechanism of the antiparkinsonian effects of 5-HT2A receptor antagonists has not been fully elucidated. We have shown that the selective 5-HT2A receptor antagonist M100907 [R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenethyl)]-4-piperidinemethanol] improved motor impairments in mice treated with the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In Parkinsons disease (PD) patients and animal models of parkinsonism dopamine denervation is associated with increased cortico-striatal glutamatergic transmission. We hypothesized that 5-HT2A receptor antagonists may exert their antiparkinsonian effects by decreasing striatal glutamate. Here, using in vivo microdialysis, we have shown an increased basal level of extracellular striatal glutamate when measured 3weeks after MPTP administration. The local administration of M100907 to the striatum significantly decreased striatal extracellular glutamate levels in MPTP-treated and saline treated mice. Basal extracellular serotonin (5-HT) levels were also elevated, whereas dopamine (DA) levels were significantly reduced in the striatum of MPTP-treated mice. Infusion of M100907 into the striatum produced no effect on dopamine or 5-HT levels. Local application of tetrodotoxin suppressed glutamate, 5-HT and DA concentrations in striatal dialysates in the presence or absence of M100907. The striatal expression of the glutamate transporter GLT1 was unchanged. However, there was an upregulation of the expression of 5-HT2A receptors in the striatum of MPTP-treated animals. Our data provide further evidence of enhanced glutamatergic neurotransmission in parkinsonism and demonstrate that blocking 5-HT2A receptors in the striatum will normalize glutamatergic neurotransmission.

Age- and duration-dependent effects of MPTP on cortical serotonin systems

It has been well established that aging is the most prominent risk factor for PD. In the MPTP mouse model which has been widely used to study PD, studies have shown that MPTP exhibits its neurotoxic effects on the dopaminergic system in an age-dependent manner. Although it is recognized the serotonergic system is impacted in PD, how aging influences serotonergic neurodegeneration in PD has not been adequately investigated. In the present studies, we examined the long-term effects of MPTP treatment on regional concentrations of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) in the striatum and prefrontal cortex (PFC). We also determined if there are differences in the age-dependent vulnerability of the monoaminergic system to MPTP. In young (3-month-old) mice, MPTP produced significant decreases in striatal DA but no changes in striatal 5-HT and NE three weeks after MPTP treatment. There was partial recovery of striatal DA concentrations 18 months later. This was accompanied by elevated striatal 5-HT. In the PFC, NE was decreased but there was complete recovery 18 months later. By contrast, we observed a long-term decrease in prefrontal 5-HT with no recovery of 5-HT concentrations 18 months after MPTP treatment. Striatal DA and NE but not 5-HT neurons exhibited age-dependent vulnerability to MPTP. Aging had no influence on the neurotoxic effects of MPTP in the PFC. Thus, there is divergence in the response of DA and 5-HT systems to MPTP neurotoxicity.