A Practical Approach, Second Edition=Ronald D. Ho.pdf

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 663at a rate to exceed initial levels within 3 h. 69 The embryo, however, remains refractory until VYSconcentrations are restored to preexposure levels. This suggests that repletion of the VYS may beinvolved in the regulation of embryonic GSH synthesis through control of the supply of cysteine(the rate limiting precursor for GSH de novo synthesis) to the embryo or through inhibitory signals.Specific rates of GSH synthesis have been determined by measurement of the specific incorporationof [ 35 S]cysteine into GSH during periods of normal embryogenesis, and the parallel useof [ 35 S]methionine to show that no cystathionine pathway exists in the conceptus during this phaseof development. 69 Although the embryo has the enzymatic capacity to replenish GSH, embryonicsynthesis is not activated until the VYS is GSH-replete, perhaps due to regulation of the precursoramino acid supply. Investigations using whole embryo culture are currently underway to understandthe regulation of GSH precursors, their supply to the embryo, and possible interruptions resultingfrom xenobiotic exposure. The mechanisms of amino acid uptake and utilization have already beenaddressed, 113–115 but a lack of information still exists regarding the perturbation of these processesby chemical agents. The in vitro whole embryo culture model will also serve well for studiesdesigned to help understand the mechanisms of GSH stimulation, induction, and response duringchemical and oxidative stress.The ability to control conditions such as the time and concentration of exposure, oxygen tension,temperature, pH, and other environmental factors makes whole embryo culture a very useful systemfor evaluation of certain endpoints. These include effects of oxidative stress, production of reactiveoxygen species (ROS), 116 and the antioxidant functions that protect the conceptus from theseconditions. Direct additions of the enzymes superoxide dismutase, catalase, and glutathione peroxidasehave all been shown to affect outward manifestations of embryotoxicity in cases of exposureto agents such as glucose (hyperglycemia), 117 arsenic, 118 and phenytoin. 119 It is not yet understood,however, what the mechanisms of action are and/or whether the enzymes are exerting their effectsintra- or extracellularly. Superoxide dismutase, catalase, glutathione disulfide reductase, glutathioneperoxidase, γ-glutamyl transpeptidase, and other related protective enzymes have been postulatedto be expressed differentially in space and time throughout the continuum of development. Thismay well be responsible for some of the selective sensitivities and resistance seen at differentjunctures in gestation. Mechanistic biochemical and molecular investigations are currently underwayto probe the possibility that the regulation of programmed cell death is a function of ROSproduction and the cells ability to maintain the proper redox and antioxidant status. 120Closely related to all of the foregoing discussions of biotransformation, oxidative stress, celldeath, DNA repair, and maintenance of the proper intracellular environment are the roles ofextracellular calcium, 121 calcium channel blockers, 122,123 and the potential susceptibility of alteredcalcium homeostasis in the dysmorphogenesis of neural tube closure. 124 Regulation and maintenanceof pyridine nucleotide [NAD(H) and NADP(H)] status during development and the critical alterationsthat occur following chemical insult have also been identified as important factors inmechanisms of teratogenesis and embryonic development, but have not yet been systematicallystudied in the developing conceptus. The status and turnover of NAD(H) in aminothiadiazoleteratogenicity, 125 possibly related to DNA repair initiated through poly-ADP ribosylation andhypoxia, 126 have been addressed using in vitro systems. An important role of the major cellularreducing equivalents, NADP(H) and NAD(H), has been postulated for a number of critical biochemicalpathways and reactions involving normal developmental metabolism and their responseto xenobiotic insult, including the endogenous production of antimetabolites. 126–128 Because of theneed to monitor very rapid changes in redox status that may be altered due to chemical orenvironmental disruption of cellular function, Thorsrud and Harris 126 have used the whole embryoculture system and noninvasive, real-time MFOP fluorescence probes. These probes were used tomeasure pyridine nucleotide fluxes from the VYS surface of intact, viable rat and mouse conceptusesmaintained in a customized perfusion chamber (Figure 16.5). Chemical modulation of antioxidantfunctions and concurrent determinations of GSH status have shown that measured changes of© 2006 by Taylor & Francis Group, LLC