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C Harris

Publications and source records attributed to C Harris.

At least 127 records · Page 7Linked to original sources
PubMed1995

Formation of protein-glutathione mixed disulfides in the developing rat conceptus following diamide treatment in vitro.

Protein-glutathione mixed disulfide (protein-S-SG) formation was investigated in developing rat conceptuses during early organogenesis (gestational day 10, GD 10) using the whole embryo culture system. Low levels of protein-S-SG (25.0 +/- 6.6 pmoles resolved GSH/conceptus) were found in conceptuses under normal culture conditions. Incubation of the conceptuses with 75-500 microM diamide (a thiol oxidant) resulted in rapid increases in protein-S-SG (to 2- to 16-fold that of control values) in a dose-dependent manner during 30 min of the culture period. Approximately 20% of the observed cytosolic glutathione (GSH) depletion following diamide (500 microM) could be accounted for as mixed disulfides of protein sulfhydryls, when determined in whole conceptual tissues after 15 min. The most extensive S-thiolation of protein sulfhydryls by GSH was observed in visceral yolk sac (VYS) when compared to embryo proper and ectoplacental cone. This result indicates that the most abundant, sensitive, or accessible protein sulfhydryls were found in the VYS. Inhibition of glutathione disulfide reductase activity by pretreatment of the conceptuses with 25 microM BCNU for 2 hr potentiated protein-S-SG formation elicited by 75 microM diamide. Reincubation of the conceptuses in fresh media, following the 15-min treatment with 500 microM diamide, reversed both the GSH depletion and the protein-S-SG formation in conceptal tissues. The reduction of the protein-S-SG was dependent on adequate intracellular GSH levels and was inhibited when GSH was rapidly depleted by subsequent addition of N-ethylmaleimide (NEM, 100 microM). Under the same experimental conditions, addition of 1 mM dithiothreitol (DTT) did not significantly enhance the GSH restoration rate nor the protein-S-SG reduction rate. The results also indicated that low levels of intracellular cysteine do not play an important role in the reduction of protein-S-SG. Protein-S-SG formation may be important for cellular regulation and in mediating the embryotoxicity elicited by diamide or other oxidative stresses.

Animals↗
PubMed1995

Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro.

Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in increased glutathione disulfide (GSSG) and protein-glutathione mixed disulfide (protein-S-SG) formation, which is accompanied by embryotoxicity and reductions in amniotic fluid volume. The altered functions of critical proteins or enzymes caused by the formation of protein-S-SG perturb cellular metabolism and may be involved in the embryotoxicity produced by GSH oxidation. The present study investigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by diamide exposure in organogenesis-stage rat conceptus (gestational day 10) in vitro. The metabolism of glucose via the PPP, measured as amounts of CO2 production from D-[1-14C]-glucose, was significantly increased in the conceptus exposed to 100-500 microM diamide to levels 2.5-3-fold those of controls. It was found that these substantial increases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway. Changes in glycolysis due to diamide treatment were also determined by measurements of lactate production from D-[U-14C]-glucose. Production of lactate by the conceptus exposed to 250-500 microM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis. Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yolk sac (VYS) compared to those from the embryo. Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when determined in the supernatants from the VYS of the conceptus exposed to 500 microM diamide for 60 min. In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N-ethylmaleimide (NEM, 100 microM) within 15 min of the exposure. In contrast to diamide and NEM, no alterations in lactate production were seen in the conceptus treated with the GSH depletor L-buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media. Further experiments demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 microM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent. These results indicated the involvement of thiol/disulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and protein-S-SG formation could be a critical event associated with mechanisms of embryotoxicity elicited by oxidative stress. It was suggested in this study that, under these experimental conditions, embryotoxicity induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis).

Animals↗
PubMed1995

Glutamate-mediated excitotoxic death of cultured striatal neurons is mediated by non-NMDA receptors.

Considerable interest has focused on the role of glutamate-mediated excitotoxicity in neurodegenerative disorders of the basal ganglia. The in vitro data on the receptor mechanisms involved in this process, however, have been inconclusive. Some studies have indicated that excitotoxins acting at NMDA receptors kill striatal neurons and others have indicated that NMDA receptor-mediated excitotoxic death of striatal neurons is minimal in the absence of cortex. In the present study, we used a pharmacological approach to carefully reexamine this issue in 2-week-old cultures of striatal neurons dissociated from E17 rat embryos. The sensitivity of these neurons to glutamate agonists and antagonists was determined by monitoring cell loss in identified regions of the growth dishes. We found that glutamate killed striatal neurons with an EC50 of 100 microM. This loss was not mediated by NMDA receptors, since it was not reduced by the NMDA receptor antagonist APV (0.1-1.0 mM). Consistent with this result, up to 50 mM NMDA receptor-specific excitotoxin quinolinic acid (QA) did not affect neuronal survival. Depolarizing the QA-exposed neurons with 35 mM potassium chloride to enhance NMDA receptor activation by QA also did not produce neuron loss. The metabotropic glutamate receptor antagonist AP3 (500 microM) also had no effect on the striatal neuron loss produced by 100 microM glutamate. In contrast, the non-NMDA antagonist GYKI 52466 (100 microM) did block the excitotoxic effect of glutamate (100 microM). Specific AMPA and KA receptor agonists and the non-NMDA antagonist GYKI 52466 revealed that the non-NMDA receptor-mediated excitotoxic effect of glutamate was mediated by KA receptors. These results suggest that cultured striatal neurons are directly vulnerable to non-NMDA glutamate agonists, but not to NMDA and metabotropic glutamate agonists. Thus, non-NMDA receptors may play a greater role in the excitotoxic death of striatal neurons in disease and experimental animal models than previously realized.

Animals↗
PubMed1995

Real time microfiberoptic redox fluorometry: modulation of the pyridine nucleotide status of the organogenesis-stage rat visceral yolk sac with cyanide and alloxan.

The surface of rat visceral yolk sacs (VYS) of intact, viable rat conceptuses were continuously monitored with a microfiberoptic sensor optimized for detection of the reduced pyridine nucleotides, NADH and NADPH. Model chemical toxins, cyanide and alloxan, were used and evaluated on the basis of their differential ability to modulate NAD(H)- and NADP(H)-dependent cellular pathways, respectively. Exposure with 2 mM sodium cyanide for 5 min caused a reversible fluorescence increase of 325 arbitrary fluorescence units (AFU) and 225 AFU on Gestational Days (GD) 10 and 11, respectively. Exposure with 40 mM alloxan for 5 min resulted in a fluorescence decrease of 170 and 120 AFU on GD 10 and 11, respectively. Glutathione (GSH) levels in the VYS, as determined by HPLC, showed a marked decrease from 27.3 +/- 2.1 to 2.9 +/- 0.4 pmol/mg protein, within the 5-min alloxan exposure period on GD 10. No decrease in GSH levels was noted for the same exposure duration on GD 11. A 2-hr pretreatment with 25 microM BCNU [(1,3 bis(2-chloroethyl)-1-nitrosourea], to inhibit glutathione disulfide reductase (GSSG-Rd), resulted in an elimination of the fluorescence decrease, but still led to a significant drop in GSH levels as seen on both days of gestation. These results are consistent with overall changes in intracellular pyridine nucleotide concentrations, where the relative amounts of NADPH increase significantly and disproportionately from GD 10 to 11. The net oxidation of NADPH, through GSSG-Rd activity, appears to be responsible for the alloxan-induced decrease in surface fluorescence. Conversely, the cyanide-induced fluorescence increases appear to be the result of NAD+ reduction, mediated through the inhibition of the terminal cytochrome oxidase in the electron transport chain.

Abnormalities, Drug-Induced↗
PubMed1994

Potential use of buccal smears for rapid diagnosis of autosomal trisomy or chromosomal sex in newborn infants using DNA probes.

Buccal smears from 3 women and 1 man were probed with alpha satellite DNA probes for chromosomes 8, 18, X, and Y. Buccal smears were also collected from an adolescent phenotypic female with uterine agenesis, as well as from newborn infants with suspected trisomy 18 and trisomy 21. The clinical cases were confirmed with conventional cytogenetic studies of peripheral lymphocytes. Overall probe efficiency at detecting expected chromosome number in interphase cells was found to be 71% +/- 6.8%. Higher than expected n-1 signal numbers may be due to karyopyknotic intermediate epithelial cells present in all collected samples. Overall probe efficiency was found to be consistent using alpha satellite and cosmid probes, both of which accurately reflected the modal copy number of the target chromosomes. False trisomy was less than 1%. This study suggests DNA probes can be used in buccal smears for rapid diagnosis of trisomies and chromosomal sex in newborns, but because of high rates of false hypoploid signals, probed buccal smear specimens may not be accurate at diagnosing mosaicism.

Adult↗
PubMed1994

Chloroquine accumulation and alterations of proteolysis and pinocytosis in the rat conceptus in vitro.

The teratogenicity of chloroquine (CQ) has been hypothesized to result from its effects on lysosomal function, specifically the ability of the visceral yolk sac (VYS) to capture and degrade external macromolecules. Using the rat whole embryo culture system, we evaluated the ability of CQ to accumulate in conceptual tissues and its effects on aspects of VYS function known to be important in the uptake and processing of nutrients. When CQ was added directly to the culture medium, it was found to accumulate rapidly in conceptual tissues, particularly the VYS. Tissue concentrations of CQ in the embryo proper reached approximately 10-fold those in the medium, whereas concentrations in the VYS exceeded by 100-fold the medium concentration within a 4-hr exposure on gestational day (GD) 10. Embryotoxic concentrations of CQ (10-30 microM) enhanced the activity of lysosomal cysteine proteinases measured in vitro under optimum pH conditions in both embryonic and VYS homogenates after a 26-hr treatment from GD 10-11. A different pattern of response in enzyme activity was observed between embryos and VYSs that could be attributed to the preferential accumulation of CQ in the VYS. Nonembryotoxic concentrations of CQ (1-7.5 microM) induced a concentration-dependent increase in VYS enzyme activity that peaked in conceptuses exposed to 20 microM CQ (an intermediate embryotoxic concentration). The enhanced cysteine proteinase activity was time dependent and appeared to increase gradually in conceptuses exposed to 10-20 microM CQ during the 26-hr culture period. This was in contrast to the rapid accumulation of CQ in conceptual tissues seen on gestational day 10. Protein content in the VYS was increased significantly after a 9-hr exposure of whole conceptuses to CQ (20 microM), indicating an inhibition of VYS proteolytic activity in situ. After 24 hr of exposure to 20 microM CQ, VYS protein content was not significantly different from control, but embryonic protein was reduced significantly by 20%. These observations are consistent with a model of reversible inhibition of VYS proteolysis by CQ followed by a compensatory increase in lysosomal proteinase activity. VYS fluid-phase pinocytosis was also assessed after CQ exposure and found to be inhibited only in the highest CQ concentration tested (30 microM). Lower concentrations of CQ that were still embryotoxic (10-20 microM) did not affect VYS fluid-phase pinocytosis, suggesting that inhibition of this activity is not primarily responsible for CQ embryotoxicity.

Animals↗
PubMed1994

In vitro embryotoxicity of the cysteine proteinase inhibitors benzyloxycarbonyl-phenylalanine-alanine-diazomethane (Z-Phe-Ala-CHN2) and benzyloxycarbonyl-phenylalanine-phenylalanine-diazomethane (Z-Phe-Phe-CHN2).

This study makes use of whole embryo culture to investigate the potential embryotoxicity of benzyloxycarbonyl-phenylalanine-alanine-diazomethane (Z-Phe-Ala-CHN2) and benzyloxycarbonyl-phenylalanine-phenylalanine-diazomethane (Z-Phe-Phe-CHN2), two low molecular weight, active site-directed and irreversible inhibitors of the lysosomal cysteine proteinases. Peptidyl diazomethanes are the most specific inhibitors available for lysosomal cysteine proteinases and can be hypothesized to interrupt visceral yolk sac (VYS)-mediated nutrition during early organogenesis. When added directly to the culture medium of gestational day 10-11 rat conceptuses, both compounds inhibited lysosomal cysteine proteinase activity in the VYS in a concentration-dependent fashion that correlated with the degree of embryotoxicity observed. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were also found to increase the protein content of the VYS, even though all other conceptual growth parameters decreased. This effect was dependent on the serum content of the culture medium and the exposure time. Histological examination of Z-Phe-Ala-CHN2-treated conceptuses revealed a dramatic increase in the size and number of vacuoles in the VYS endoderm epithelium, suggestive of inhibition of VYS proteolysis. At the same time, excessive cell death was observed throughout the neuroepithelium and in specific regions of the mesenchyme of the corresponding embryos. This cell death manifested morphological characteristics of apoptosis and could be detected by supravital staining with Nile Blue Sulphate. These findings provide additional evidence in support of the hypothesis that lysosomal cysteine proteinases play a critical role in VYS-mediated histiotrophic nutrition and suggest that peptidyl diazomethanes may be useful in further characterization of these enzymes. The possible direct effects of these inhibitors on embryonic cells and the relationships between interruption of VYS-mediated nutritional processes and embryonic cell death are discussed.

Animals↗
PubMed1994

Female reproductive and developmental toxicology: overview and current approaches.

In recent years, concern about possible female reproductive and developmental toxicity due to environmental contaminants, such as PCBs, has been growing. Because this area of toxicology had not been emphasized prior to this time, there are many gaps in current knowledge about female developmental and reproductive toxicology and only a limited number of validated tests to assay effects of toxicants on various parts of the reproductive and developmental cycle. This article reviews the current state of knowledge on this topic and also explores a variety of techniques for assessing female reproductive and developmental toxicity. These include an assay of the state of intercellular communication among the embryo, fetus and placenta; protocols for assessing toxicity in early pregnancy; and techniques for evaluating the role of glutathione in protecting the conceptus from xenobiotics.

Embryonic and Fetal Development↗
PubMed1994

Sodium channel mutations in acetazolamide-responsive myotonia congenita, paramyotonia congenita, and hyperkalemic periodic paralysis.

Hyperkalemic periodic paralysis (hyperKPP) and paramyotonia congenita (PC) are genetic muscle disorders sharing the common features of myotonia and episodic weakness. In hyperKPP, patient symptoms and signs are worsened by elevated serum potassium, whereas in PC, muscle cooling exacerbates the condition. There are patients in whom features of both hyperKPP and PC are present. These diseases result from molecular alterations in the adult skeletal muscle sodium channel. This report summarizes our sodium channel mutation analysis in 25 families with hyperKPP and PC. We also report the putative disease-causing mutation in acetazolamide-responsive myotonia congenita, a related disease in which myotonia is worsened by potassium but in which episodic weakness does not occur. This missense mutation (I1160V) occurs at a very highly conserved position in the sodium channel, cosegregates with the disease, and was not present in any of a large panel of normal DNAs. Electrophysiologic characterization of specific mutations will lead to better understanding of the biophysics of this voltage-gated ion channel.

Acetazolamide↗
PubMed1994

Severity of articular cartilage abnormality in patients with osteoarthritis: evaluation with fast spin-echo MR vs arthroscopy.

OBJECTIVE: The purpose of this study was to assess the accuracy of fast spin-echo MR imaging for depicting the severity of articular cartilage abnormalities in patients with osteoarthritis. SUBJECTS AND METHODS: Twenty-three subjects (10 volunteers less than 35 years old and 13 patients with proved, symptomatic, idiopathic osteoarthritis of the knee of 6 months' to 10 years' clinical duration) underwent fast spin-echo MR imaging of the knee. Two observers graded each articular surface using a five-category scale that took into account abnormalities in the signal intensity of cartilage as well as thickness and contour. The 13 patients also underwent arthroscopic evaluation (as part of a separate protocol) in which cartilage abnormalities were graded by using a similar five-category grading scale, without the graders knowing the results of MR imaging. Articular cartilage was assumed to be normal in the volunteers. RESULTS: One hundred thirty-seven joint surfaces were graded; one surface was obscured by artifact and was excluded. The Spearman rank linear correlation between arthroscopic and MR grading was highly significant (p < .002) for each of the six articular regions evaluated. The MR and arthroscopic grades were the same in 93 (68%) of 137 joint surfaces, they were the same or differed by one grade in 123 surfaces (90%), and they were the same or differed by one or two grades in 129 surfaces (94%). CONCLUSION: Our results suggest that fat-presaturated fast spin-echo MR imaging depicts the severity of articular cartilage abnormalities in osteoarthritis with reasonable accuracy, as compared with arthroscopic evaluation as the standard of reference.

Adult↗
PubMed1994

Alterations in oxygen consumption during and between bouts of level and downhill running.

Since the etiology of the drift in VO2 during downhill running is unclear, this study was designed to assess the contribution of heart rate (HR), ventilation (VE), blood lactate, rectal temperature (RT), muscle damage, and several variables that have not previously been included in VO2 drift research: muscle temperature (MT), and stride rate (SR) and length (SL), to the drift in VO2. Six subjects participated in a 45-min level run (LEVEL) and two 45-min downhill runs (DOWN1 and DOWN2) at 50% VO2max. Although VO2 increased significantly over time for all bouts, the magnitude [4.3% (LEVEL), 5.4% (DOWN1), and 8.1% (DOWN2)] did not differ between bouts (P > 0.05). VO2 was significantly lower during DOWN2 than during LEVEL and DOWN1 (P < 0.05). MT increased during the three bouts (P < 0.05) but the change over time was not different between bouts. SR and SL did not change over time within each bout nor between the two downhill runs. Muscle damage, as indicated by serum creatine kinase levels and perceived soreness, was less following LEVEL and DOWN2 than DOWN1 (P < 0.05). HR and RT increased over time (P < 0.05) but did not differ between bouts. VE and blood lactate did not differ over time or between bouts. VO2 drift during the three bouts paralleled changes in RT, MT, and HR but appears unrelated to muscle damage or biomechanical factors.

Adult↗
PubMed1994

Comparison of aminobenzoate potassium and placebo in the treatment of scleroderma.

OBJECTIVE: To determine the safety and efficacy of aminobenzoate potassium (KPAB) in treating the skin manifestations of scleroderma. METHODS: Via a 48-week prospective, randomized, double blind, placebo controlled trial we compared the efficacy of KPAB 12 g/day with matching placebo. Outcome measures included skin mobility and thickening scores, patient and physician global assessments and, measurements of maximal oral aperture and hand range of motion. RESULTS: Of 146 patients who entered the study, 76 (52%) completed. Demographics of the study population included age 49 +/- 13 years, 83% women, mean (range) disease duration was 104 (7-600) months. There were no differences in the demographics of the KPAB vs placebo nor the group that completed the study compared with the withdrawal group. There were no clinical or statistically significant differences between the KPAB and the placebo treated groups in any of the outcome measures. Subgroup analyses of skin mobility and skin thickening based on age, extent of disease, severity of disease, duration of disease and involved vs uninvolved skin were performed, but no differences were noted. The overall compliance to the medical regimen was > or = 75% in 93% of patients completing the study. Eighteen patients in the KPAB group and 6 placebo patients withdrew due to adverse drug reactions (ADR). The most common withdrawals for ADR were gastrointestinal intolerance and headaches. All ADR resolved following withdrawal of medication. CONCLUSION: KPAB did not alter the skin changes of scleroderma in a group of patients with relatively longstanding stable disease. KPAB was reasonably well tolerated in this group of patients.

4-Aminobenzoic Acid↗
PubMed1993

p53 mutation is associated with progression in follicular lymphomas.

The majority of low-grade follicular lymphomas will eventually transform to an aggressive intermediate, or high-grade lymphoma. The molecular mechanisms responsible for this transformation have not been determined. We studied serial biopsies from 34 patients with follicular lymphomas that underwent histologic transformation, for abnormalities of the p53 tumor suppressor gene by a combination of immunohistochemistry, single strand conformation polymorphism analysis (SSCP), and sequencing. We found overexpression of p53 in 10 of the 34 transformed aggressive lymphomas, 9 of which contained mutations identified by SSCP analysis and subsequent sequencing. Matched pretransformation low-grade follicular lymphoma biopsies were available for 7 of the 10 cases. None of six studied by immunohistochemistry showed overexpression of p53 and only 1 of 4 studied by SSCP/sequencing showed the presence of mutation in the pretransformation biopsy. Interestingly, an eighth p53 positive transformed lymphoma recurred with a clonally related, p53 negative low-grade lymphoma 5 years after the patient had achieved a complete remission. Immunohistochemistry also showed that several pretransformation biopsies from p53 positive transformed cases showed rare p53 positive cells and in one case we could document an increase in their number over time. Twenty-five additional low-grade follicular lymphoma biopsies were also examined. Three patients had lymphomas positive for p53 mutation. One of the three subsequently transformed within a year of the biopsy studied; the second patient had an earlier (unavailable) biopsy at a different site that showed transformed histology. The third patient was treated with ProMACE-MOPP combination chemotherapy and attained a complete remission. We conclude that (1) mutations of p53 are associated with histologic transformation in approximately 25% to 30% of follicular lymphomas and (2) p53 positive cells can be detected before histologic transformation, but do not comprise a significant percentage of the neoplastic cell population (identifiable by SSCP) until late in the disease, just before or after histologic progression. Finally, the data also suggest that p53 positive low-grade lymphomas are at risk for progression and that in this subset, aggressive therapy may be warranted.

Base Sequence↗
PubMed1993

Chloroquine embryotoxicity in the postimplantation rat conceptus in vitro.

The embryotoxicity of the antimalarial drug chloroquine (CQ) was evaluated in vitro using the rat whole embryo culture system. CQ was found to be embryotoxic and dysmorphogenic when added directly to the culture media containing gestational day (GD) 10 rat conceptuses. Twenty-six-hr exposure to CQ elicited dose-related decreases in embryonic crown-rump length, protein and DNA contents and increases in the incidence of morphologically abnormal embryos. At 30 microM CQ, embryonic protein content was decreased to 67% and DNA content to 58% of control while the incidence of morphological abnormalities rose to 100%. Abnormal axial rotation, micro-ophthalmia, and selective cephalic hypoplasia were the most common developmental abnormalities observed. Visceral yolk sac (VYS) vasculature and blood pigmentation were also decreased in a dose-dependent manner, as was VYS DNA content (80% of control at 30 microM). VYS protein content, however, showed an alternate pattern of response, decreasing to 87% of control at 10 microM CQ but increasing to 125% of control at 30 microM. Histologic evaluation revealed that the cytoplasm of the VYS endoderm epithelium was distended due to vacuolization produced by CQ exposure. In the embryo proper, CQ inhibited cranial neural tube development and altered the morphology of cranial neural crest cells. These observations document the in vitro embryotoxicity of CQ and suggest altered VYS histiotrophic nutrition as well as direct embryonic effects as possible mechanisms of CQ embryotoxicity.

Abnormalities, Multiple↗
PubMed1993

Real time micro-fiberoptic monitoring of endogenous fluorescence in the rat conceptus during hypoxia.

A micro-fiberoptic methodology has been developed for non-invasive, real time measurement of endogenous pyridine nucleotide fluorescence from the surface of the visceral yolk sac (VYS) in intact, viable rat conceptuses. Gestational day (GD) 10-12 conceptuses are maintained in a customized perifusion system, which allows for control of oxygenation, as well as the continuous measurement of pH and oxygen concentration in the effluent perifusate. Miniaturized light guides were constructed by drawing 250 microns ESKA acrylic optical fibers through a stainless steel sheath with a high strength epoxy polymer. A single fiber supplied the excitation signal from a mercury arc lamp at a wavelength of 366 nm. The emission signal was returned via three additional fibers, electronically amplified, processed, and recorded, using a dual channel lamp-compensated fluorometer, optimized for detection of reduced pyridine nucleotides at 455 nm. Endogenous fluorescence in the conceptus was monitored by placing the polished tip of the sensor directly on the surface of the VYS. Oxygen-equilibrated conceptuses, exposed to 100% nitrogen, produced a reproducible biphasic surface fluorescence peak, which returned to baseline levels upon reoxygenation of the perifusate. This biphasic response consisted of an initial rapid rise in fluorescence (phase I), followed by an attenuated rate in fluorescence signal increase (phase II). The hypoxia produced age-dependent rates of fluorescence change during phase I, while phase II remained relatively unchanged throughout GD 10-12. These results demonstrate the ability to monitor endogenous fluorescence, non-invasively and in real time, during the period of organogenesis in the intact rat conceptus and will provide valuable information in studies of embryonic metabolism and response to chemical embryotoxicants.

Animals↗