TOXICOLOGY AND APPLIED PHARMACOLOGY 92,170-l 78 ( 1988) Role of the 4-Hydroxy Intermediate in the in Vitro Embryotoxicity of Cyclophosphamide and Dechlorocyclophosphamide VALERIE L. SLOTT AND BARBARA F. HALES Department of Pharmacology & Therapeutics and Centre for the Study of Reproduction, McGill University, 3655 Drummond Street, Montreal, Quebec H3G I Y6. Canada Received May 15, I98 7; accepted October 5, 198 7 Role of the 4-Hydroxy Intermediate in the in Vitro Embryotoxicity of Cyclophosphamide and Dechlorocyclophosphamide. SLOTT, V. L., AND HALES, B. F. (1988). Toxicol. Appl. Phar- macol. 92, 170-I 78. Cyclophosphamide must be metabolically activated to produce malforma- tions in cultured rat embryos. A 4-hydroxylated intermediate, 4-hydroxycyclophosphamide is initially formed during this activation. While 4-hydroxycyclophosphamide (and/or its open-ring tautomer. aldophosphamide) is believed to act as a transport form in mediating the antineoplas- tic activity of cyclophosphamide, its role in the teratogenicity of this drug is not known. In this study the effects of two “preactivated” cyclophosphamide analogs on cultured Day 10 rat em- bryos were determined. The first analog, 4-hydroperoxycyclophosphamide, is converted to 4- hydroxycyclophosphamide in aqueous solutions, releasing both acrolein and phosphoramide mustard, while the second, 4-hydroperoxydechlorocyclophosphamide, releases, in a similar manner, acrolein and the inactive metabolite, phosphoric acid diamide. Both cyclophospha- mide analogs were teratogenic, embryolethal, and growth retarding in vitro, but the effective concentrations and the types of malformations produced were different. 4-Hydroperoxycyclo- phosphamide produced embryo deaths and malformations and decreases in embryonic growth and protein content at concentrations in the range of 5 to 25 .uM. In contrast, 4-hydroperoxy- dechlorocyclophosphamide did not produce embryo deaths at concentrations below 100 pM and produced embryo malformations and growth retardation only at 125 pM. The concentration- response curve and the spectrum of malformations produced by 4-hydroperoxycyclophospha- mide resembled those previously reported for phosphoramide mustard, while the concentra- tion-response curve and types of malformations produced by 4-hydroperoxydechloro- cyclophosphamide more closely resembled those observed with acrolein. Thus, the 4-hydroxy intermediates are similar as teratogens to the most potent ofthe metabolites which they produce: the 4-hydroxy compounds may serve as a transport form ofcyclophosphamide but do not appear themselves to have a major role in teratogenicity. 0 1988 Academic Press. Inc. The antineoplastic and immunosuppressant phosphamide; 4-hydroxycyclophosphamide drug cyclophosphamide is teratogenic in a equilibrates with its open-ringed tautomer, variety of species (Chaube et al., 1967; Ger- aldophosphamide, which then undergoes linger, 1964; Gibson and Becker, 1968; spontaneous P-elimination to yield two cyto- Greenberg and Tanaka, 1964; McClure et al., toxic metabolites, phosphoramide mustard 1979). It is now well documented that cyclo- and acrolein. The identity of the circulating phosphamide requires metabolic activation metabolite that enters cells and ultimately before it can have therapeutic effects (Brock gives rise to the oncotoxicity is still controver- et al., 1971; Foley et al., 1961). The first step sial, but most investigators favor 4-hydroxy- in the activation of cyclophosphamide is 4- cyclophosphamide/aldophosphamide for hydroxylation of the oxazophosphorine ring this role (Brock, 1976; Brock and Hohorst, to form the intermediate, 4-hydroxycyclo- 1977; Cox et al., 1977; Domeyer and Sladek, 0041-008X/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights ofreproduction in any form reserved. 170