Identification and quantification of cocaine N-oxide: a thermally labile metabolite of cocaine.
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In this article, we report the identification and quantitation of cocaine N-oxide (CNO), a thermally labile oxidative metabolite, from both animal and human samples. The concentration of CNO is similar to the concentrations of cocaine in the samples analyzed. The technique used for the determination of CNO in this study is liquid chromatography-electrospray ionization mass spectrometry, which is necessary because CNO is converted to cocaine upon heating. This includes simple heating of aqueous solutions to temperatures in excess of 100 degrees C and analysis by gas chromatography-mass spectrometry (GC-MS), in which CNO is converted to cocaine in the injection port. The thermal conversion of CNO to cocaine is estimated to cause an over-reporting of cocaine levels by 10-20% when using GC-MS. (+info)
Novel insights into human endometrial paracrinology and embryo-maternal communication by intrauterine microdialysis.
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The regulation of human implantation is still unknown. Evidence from mice suggests an essential role for several paracrine mediators but species differences with implantation in the human preclude the extrapolation of these concepts to humans. An intrauterine microdialysis device (IUMD), consisting of microdialysis tubing glued into a balloon catheter on one side and into a polypropylene tube on the other, allows a dynamic and accurate in-vivo measurement of uterine paracrine interactions in humans. Inserted into the uterine cavity in the form of a loop, it can be continuously perfused with saline to reveal a number of relevant cytokines and growth factors in uterine effluents of non-pregnant women in both follicular and luteal phases. These included interleukin (IL)-1alpha, IL-1beta, IL-6, leukaemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), epidermal growth factor, vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-1 (IGFBP-1), prolactin, and human chorionic gonadotrophin (HCG). The source of intrauterine HCG is unclear since endometrial mRNA for the HCG beta-subunit is not revealed using reverse transcriptase polymerase chain reaction analysis. Applying urinary HCG locally via the IUMD profoundly alters endometrial secretory parameters. Prolactin, IGFBP-1, and M-CSF are significantly inhibited and VEGF is regulated in a biphasic manner involving early stimulation followed by inhibition of intrauterine levels. Use of the IUMD has thus shown that the urinary HCG preparations routinely used for ovulation induction and luteal support may directly alter endometrial function. (+info)
Maternal adrenocortical hormones maintain the early development of pancreatic B cells in the fetal rat.
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To investigate the effect of maternal adrenocortical hormones on the development of fetal pancreatic islet cells, pregnant rats were adrenalectomised on d 6 of gestation. On d 12-16 the growth patterns of fetal insulin-producing B cells, glucagon-producing A cells, and somatostatin-producing D cells were observed histometrically. Maternal adrenalectomy resulted in growth retardation of fetal B cells on d 12-15. Maternal corticosterone therapy prevented this retardation. Maternal adrenalectomy, however, did not affect the developmental patterns of A and D cells. By Western blotting and immunohistochemistry, glucocorticoid receptors were demonstrated to be present in the islet cells from d 12 to d 15. These results suggest that maternal adrenocortical hormones, glucocorticoids in particular, maintain the early development of fetal pancreatic B cells through their specific intracellular glucocorticoid receptor. (+info)
RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.
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C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development. (+info)
Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice.
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The aryl hydrocarbon receptor (AhR) mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and transcriptional activation of genes encoding a number of xenobiotic metabolizing enzymes. Prenatal exposure of mice to TCDD causes severe alterations in embryo and fetal development, including hydronephrosis and cleft palate. However, the mechanisms underlying these effects are unclear. In this work, the teratogenicity of TCDD in AhR-null mice was evaluated to determine if this effect is mediated by the AhR. Homozygous wild-type (+/+) or AhR-null (-/-) female mice were mated with males of the same genotype overnight. On gestation day (GD)-10, mice were intubated orally with either corn oil (vehicle control) or 25 micrograms/kg TCDD. Fetuses were examined on GD18 for visceral and skeletal alterations. For non-TCDD-exposed litters, all developmental endpoints were comparable between genotypes, with the exception of a lower incidence of large interfrontal bones in (-/-) mice. For TCDD-exposed litters, (+/+) fetuses had a significantly greater incidence of cleft palate, hydronephrosis, small kidneys, tortuous ureters and greater dilation of the renal pelves and ureters compared to (-/-) fetuses. Interestingly, an increased resorption rate was observed in (-/-) fetuses exposed to TCDD. Results from this work demonstrate that fetal development per se is generally unaffected by the absence of the AhR or that other genes may have compensated for the loss of the AhR. More importantly, these data indicate that the AhR mediates TCDD-induced teratogenicity. Further, since a higher percentage of resorptions was observed in (-/-) litters from TCDD-treated dams, it is possible that AhR-independent mechanisms contribute to TCDD-induced developmental toxicity. (+info)
The CTLA-4 gene is expressed in placental fibroblasts.
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In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation. (+info)
Experimentally induced bovine spongiform encephalopathy did not transmit via goat embryos.
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Goats are susceptible to experimental challenge with bovine spongiform encephalopathy (BSE). This study set out to investigate whether the transmission of BSE could occur in goats following the transfer of embryos from experimentally infected donor females into uninfected recipient females. The results showed no evidence of transmissible spongiform encephalopathy disease in any of the offspring which developed from embryos from infected donors, nor indeed in any of the recipient females used as surrogate dams. In addition, there was no indication of experimental BSE spreading as either a venereal infection to males used in mating or by maternal transmission to offspring born naturally to experimentally infected donors, although numbers were small. (+info)
Comparison of urinary 6beta-hydroxycortisol/cortisol ratio between neonates and their mothers.
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AIMS: To assess CYP3A enzyme activity in human neonates by measuring the urinary 6beta-hydroxycortisol/cortisol (6beta-OHF/C) ratio. METHODS: Fifty-six mature male neonates with normal delivery, seventeen of their mothers and twenty-four healthy non-pregnant young women participated in this study. Urinary 6beta-OHF/C ratio was determined on the day of birth in neonates and their mothers. In addition, changes in the ratio after birth were determined in neonates. RESULTS: On the day of birth, the urinary 6beta-OHF/C ratio of neonates was significantly higher than that of their mothers (20.5 vs 6.9). In contrast, no significant difference was observed in the mean ratio of urinary 6beta-OHF/C between women with and without pregnancy (6.9 vs 9.0). The urinary 6beta-OHF/C ratio after birth was decreased day by day in neonates. CONCLUSION: These results indicate that the high urinary 6beta-OHF/C ratio in mature neonates on the day of birth is independent of the activity of CYP3A enzyme in their mothers. (+info)