Teratogenesis, i.e., the genesis of birth defects, is a complex process and one basic aspect of this process is teratogen-mediated cell death. More significant is the observation that teratogens kill certain cells in the embryo but spare others, i.e., teratogens exhibit cell-specific cytotoxicity. The long-term objectives of the proposed research are to understand the basis for cell spcific cytotoxicity and its relationship to the overall process of teratogenesis. Using the technique of in vitro culture of mammalian (rat) embryos during the early phases of organogenesis, we have shown that the well-studied teratogen, cyclophosphamide (CP), induces a cytotoxic response in certain cells of the embryonic head. Cells of the developing heart, however, are completely resistant to the cytotoxic effects of CP. Building upon this information, we have developed a model system for studying the mechanisms underlying this differential sensitivity. Hopefully, information gained studying this model system will provide insights into the more general problem of why some cells within a developing tissue are sensitive to the cytotoxic effects of teratogens while others are resistant.
The specific aims of this continuation proposal are to test the following hypotheses. First, cell-specific cytotoxicity is related to the production of specific types of DNA damage, i.e., DNA single strand breaks, DNA-DNA crosslinking, and DNA-protein cross linking by CP, more specifically the active metabolites of CP: phosphoramide mustard (PM) and acrolein (AC). This will be accomplished using the technique of alkaline elution. We will also investigate two factors which may modulate the interaction of PM and/or AC with embryo DNA, i.e., interactions with cellular proteins and conjugation with glutathione. Second, cell-specific cytotoxicity is related to repair of DNA damage. We will use alkaline elution and teratogen-DNA adduct antibodies to follow DNA repair. Third, cell-specific cytotoxicity is related to altered transcription. The techniques of CsTFA density gradient centrifugation, affinity chromotography, and cDNA probes coupled with in situ hybridization will be used. Fourth, cell-specific cytotoxicity is related to interactions between CP (PM and AC) and embryo RNA. In this analysis we will use the procedures of in vitro translation of modified and unmodified RNA and a two-dimensional gel electrophoretic analysis of translation products.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Human Embryology and Development Subcommittee 2 (HED)
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University of Washington
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Bratton, S L; Jardine, D S; Mirkes, P E (1997) Constitutive synthesis of heat shock protein (72 kD) in human peripheral blood mononuclear cells: implications for use as a clinical test of recent thermal stress. Int J Hyperthermia 13:157-68
Cunningham, M L; Mac Auley, A; Mirkes, P E (1994) From gastrulation to neurulation: transition in retinoic acid sensitivity identifies distinct stages of neural patterning in the rat. Dev Dyn 200:227-41
Mirkes, P E; Doggett, B; Cornel, L (1994) Induction of a heat shock response (HSP 72) in rat embryos exposed to selected chemical teratogens. Teratology 49:135-42
Mac Auley, A; Werb, Z; Mirkes, P E (1993) Characterization of the unusually rapid cell cycles during rat gastrulation. Development 117:873-83
Mirkes, P E; Doggett, B (1992) Accumulation of heat shock protein 72 (hsp 72) in postimplantation rat embryos after exposure to various periods of hyperthermia (40 degrees -43 degrees C) in vitro: evidence that heat shock protein 72 is a biomarker of heat-induced embryotoxicity. Teratology 46:301-9
Little, S A; Mirkes, P E (1992) Effects of 4-hydroperoxycyclophosphamide (4-OOH-CP) and 4-hydroperoxydechlorocyclophosphamide (4-OOH-deCICP) on the cell cycle of post implantation rat embryos. Teratology 45:163-73
Mirkes, P E; Brown, N A; Kajbaf, M et al. (1992) Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide. Chem Res Toxicol 5:382-5
Mirkes, P E; Cornel, L (1992) A comparison of sodium arsenite- and hyperthermia-induced stress responses and abnormal development in cultured postimplantation rat embryos. Teratology 46:251-9
Harris, C; Juchau, M R; Mirkes, P E (1991) Role of glutathione and hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos. Teratology 43:229-39
Mirkes, P E; Ellison, A; Little, S A (1991) Role of aldehyde dehydrogenase (ALDH) in the detoxication of cyclophosphamide (CP) in rat embryos. Adv Exp Med Biol 284:85-95

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