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

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 679similar to their behavior in the intact organism. Some of the first successful attempts at clonalanalysis to demonstrate similar behavior in vivo and in vitro involved the culture of quail myoblastsfor which various degrees of success were obtained, depending on the substrate and the constituentsof the culture medium used. 244 A number of cell types can now be dissociated from the intactorganism, conceptus, organ, or tissue, and successfully grown in culture, where the proper choicesof medium and substrate are still the critical components in determining success. Techniques forprimary culture have been established for heart myoblasts, 245 chondrocytes, 246 cartilage, 247 pigmentedretina, 248 lens, 249 and cranial neural crest. 250,251Use of primary embryonic cell cultures and established cell lines in toxicology usually focuseson proliferation or on the progression of differentiation into a specific cell type, but few haveaddressed the effects of teratogens on embryonic stem (ES) cells. Since ES cells are pluripotent,effects of specific toxicants may perturb signal transduction pathways involved in specific cell fates,resulting in the promotion of cell death, decreased proliferation, or faulty differentiation. In anycase, such effects could result in severe birth defects and embryotoxicity. ES cells have been usedto study PCB toxicity in neurogenesis. Endosulfan and 2,2′,4,4′,5,5′-hexachlorobiphenyl (2,4,5-HCB) both caused an inhibition of neurite formation in a dose-dependent manner and decreasedintracellular gap junctions and connexin 43 expression, implicating intracellular communication inearly neurogenesis. 252 The use of ES cells in mechanistic studies may serve as a more appropriatein vitro model of teratogenesis than other established cell lines. Measurement could be assessedby morphologic, biochemical, or genetic markers following toxicant exposure. These approachescould yield a great deal of information about sensitive pathways of toxicant-induced dysmorphogenesisas well as highlight the role of these pathways in development.Hypotheses relating to the proposed mechanisms of several known teratogens have implied thatalterations in cranial neural crest cell (CNC) migration, differentiation, and function are probablecauses for observed developmental anomalies. The clonal primary culture of CNC was originallydescribed in Japanese quail, using procedures such as those reported by Loring et al. 250 andGlimelius and Weston. 251 With this method, the distal portion of the trunk (containing the last 8 to10 somites) is dissected free. It is then exposed to mild enzymatic digestion (pancreatin) to loosentissue adhesions and allow the neural tube to be physically pulled free from the notochord andectoderm. Additional treatments (trypsin, collagenase) may then be needed to remove other adherenttissues before placing the neural tubes in media-filled plastic or collagen-coated petri dishes forculture. Various culture media have been used successfully, although, all require an appropriateamount of serum, 10-d chick embryo extract, and glutamine supplementation for optimal growthand differentiation. The relative proportions of serum and chick embryo extract affect both cellproliferation and cell cluster formation. This methodology takes advantage of the inherent highproliferative and rapid migratory properties of CNC cells and produces a very homogeneouspopulation that will respond to a number of chemical and environmental stimuli (Figure 16.10).Chemical agents, such as the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA),have been shown to promote cell proliferation while causing significant delays in differentiation,as manifested by alterations in melanocyte production. 251 Davis et al. 253–255 utilized the avian CNCculture system to investigate the role of free radical production and oxidative stress in the cellularpathogenesis of ethanol (EtOH) and retinoid (isotretinoin and 4-oxo-isotretinoin)-induced craniofacialabnormalities. They showed that treatment of CNC with these agents in vitro is accompaniedby the significant generation of reactive oxygen species, producing cellular blebs, loss of normalCa 2+ homeostasis, and decreased viability.This system allows investigators the flexibility to measure multiple biochemical and morphologicalendpoints and to alter several environmental conditions to probe mechanisms of ROSgeneration and cellular response. Using these approaches, Davis et al. 254 determined the CNC tobe devoid of superoxide dismutase and catalase, two important antioxidant enzymes critical forelimination of ROS. Addition of these enzymes directly to the culture medium provided significantprotection against ROS-mediated damage. In addition, those investigators were able to characterize© 2006 by Taylor & Francis Group, LLC