Relationship of DNA damage and embryotoxicity induced by 4-hydroperoxydechlorocyclophosphamide in postimplantation rat embryos.
4-Hydroperoxydechlorocyclophosphamide (4-OOHdeCl-CP) is a preactivated analogue of cyclophosphamide (CP) that undergoes an elimination reaction to yield acrolein and the nonalkylating derivative of phosphoramide mustard (PM), i.e., dechlorophosphoramide mustard. We used this analogue to assess the role of acrolein in CP-induced embryotoxicity. Embryotoxicity was assessed using day 10 rat embryos cultured in vitro. 4-OOHdeC1-CP was embryotoxic over a concentration range of approximately 75-150 microM and produced complete embryolethality at concentrations of 175 microM and above. This analogue induced abnormal development characterized by tail defects at low drug concentrations and microencephaly or prosencephalic hypoplasia at high concentrations. Using the technique of alkaline elution, we also assessed DNA damage induced by embryotoxic concentrations of drug. When embryos were cultured in serum-containing medium during drug exposure, no DNA damage was detected, even at embryolethal drug concentrations. However, if cellular glutathione (GSH) was depleted with buthionine sulfoximine (BSO) before drug exposure and embryos were cultured in serum-free medium during drug exposure, DNA damage, primarily DNA single-strand breaks, was detected, but only at embryolethal concentrations. Using radiolabeled CP, we showed that acrolein does reach the embryo; however, more acrolein is incorporated into the yolk sac. Binding studies revealed that acrolein binds preferentially to cellular protein, whereas PM binds preferentially to DNA. These results suggest that, unlike the case with PM, the embryotoxic target for acrolein is protein and not DNA. Furthermore, our results indicate that acrolein may mediate its effects on the embryo via the yolk sac.
Little
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SA
Department of Pediatrics RD-20, University of Washington, Seattle 98195.
Mirkes
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PE
D000171:Acrolein / Q000494:pharmacology / Q000633:toxicity; D000818:Animals; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D010064:Embryo Implantation; D004622:Embryo, Mammalian / Q000187:drug effects*; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D010752:Phosphoramide Mustards / Q000633:toxicity; D051381:Rats; D011919:Rats, Inbred Strains
D010752:Phosphoramide Mustards; C012358:4-hydroxycyclophosphamide; C063555:4-hydroperoxydechlorocyclophosphamide; D000171:Acrolein; D003520:Cyclophosphamide; D005978:Glutathione