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

672 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONchick has also been used as a model for classic studies in teratology. Incubation of eggs with 6-amino-nicotinamide, 192 retinoic acid, 193,194 and other agents has provided fundamental knowledgeof developmental mechanisms and their perturbation with chemical agents. More basic studies,including exposure of chick embryos to ethyl alcohol and retinoic acid derivatives, followed by anevaluation of the function and behavior of cranial neural crest cells, have looked at mechanismsinvolving specific cell types and related processes. Coupled with use of in vitro cranial neural crestcell cultures, this approach has provided considerable information showing that the neural crestmay be sensitive to chemical agents that produce or increase the production of reactive oxygenspecies. This sensitivity may be due to deficiencies in intrinsic detoxication capacity, particularlyinvolving superoxide dismutase. In many respects, analogous studies can be conducted for earlypostimplantation embryogenesis in both avian and rodent species. The chick model has the distinctadvantage, however, of being able to continue the developmental process through hatching andsubsequent maturation.In general, most nonmammalian culture systems have numerous advantages, giving them greatpotential, which, unfortunately, has not been fully utilized in the study of toxicological mechanisms.When the genomes of these organisms are completely or nearly completely mapped, their utilizationfor mechanistic studies should increase. They may eventually be fashioned into screening systemsof choice, providing useful information regarding developmental toxicity mechanisms.D. Organ and Tissue CultureOrgan culture and tissue culture are terms often used interchangeably to describe the in vitro growthof structurally and functionally intact subsets of a complete organism. The levels of biologicalorganization encompassed by this definition range from intact, functional, normally vascularizedcomplex organs to essentially avascular, relatively homogeneous tissue fragments. Organ cultureprovides numerous advantages for the study of mechanisms in developmental toxicity. It allowsfor the maintenance of a functional unit of an organism in a controlled environment, while retainingan intact architectural structure and without the need of perfusion via an intact vasculature. 196,197Specific functional and morphological processes carried out by many organ systems are influencedby chemical and physiological factors produced endogenously and also those provided by otherorgans and tissues. The roles and functions of these factors can be evaluated by their selectiveaddition or removal from the culture medium.Successful elucidation of mechanisms of toxicity produced through chemical interactions ultimatelyrequires a system in which the temporal and spatial relationships of morphological, biochemical,and molecular alterations can be evaluated. This approach necessitates the removal oftissues and organs from confounding influences, so that their interactions can be identified andcharacterized. Several embryonic organs can now be dissected free and grown successfully inculture, still meeting a number of selected experimental criteria. These include maintenance ofsimilarities in growth and differentiation, compared with in vivo patterns, and retention of the timedsequence of production of organ-specific products. These techniques are useful in manipulating thesystem to probe mechanisms and are aided by the ability to determine the exact time and durationof exposure, the precise stage of development at the time of exposure, and the degree of chemicalmetabolism prior to reaching organ targets.Organs and tissues that have been used successfully in in vitro culture applications for the studyof mechanisms of developmental toxicity include limb buds, limbs, bone, heart, palatal shelves,visceral yolk sac, eyes, sex organs, and embryonic tooth germs. Many of the initial culture techniqueswere based on Trowell’s method, 198 where the organ is grown attached to a membrane in ahumidified atmosphere. Many of these techniques have been subsequently modified to use submergedcultures and a roller apparatus in defined media to improve morphological and biochemicalfidelity in comparison with parallel in vivo developmental events (Figure 16.8). Cocultures withother organs or cells, dissociation of specific cell types from the organ, and the use of nontoxic© 2006 by Taylor & Francis Group, LLC