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© 1990 Oxford University Press

research-article

The in vitro Activation of Cyclophosphamide in the Hydra Developmental Toxicology Assay

L. M. NEWMAN, E. M. JOHNSON, R. L. GIACOBBE and L-J. Fu

Department of Anatomy. Jefferson Medical College, Thomas Jefferson University Philadelphia, Pennsylvania 19107

Received December 11, 1989; accepted May 14, 1990

The in vitro Activation of Cyclophosphamide in the Hydra Developmental Toxicology Assay. Newman, L. M., JOHNSON, E. M., GIACOBBE, R. L., AND FU, L-J. (1990). Fundam. Appl. Toxicol. 15, 488–499. The proteratogen cyclophosphamide (CP) was tested in the Hydra Assay in the presence and absence of an in vitro metabolic activation package (MAP) consisting of rat hepatic microsomes (0.06 nmol P450/ml), 500 µM NADPH, and 25 µm MgCl. This metabolic system was developed through a series of interrelated biochemical and biological assays to provide maximum cytochrome P450 mixed-function monooxygenase (MFO) metabolic capacity while controlling the inherent toxicity of the hepatic preparation and the attendant cofactors. Bioactivation of CP was confirmed under standard hydra assay conditions of pH 7.0 and 20°C and compared with activation at 37°C. Estimation of total metabolic capacity and verification of activation were made through the appearance of alkylating metabolites both in the absence and in the presence of hydra. Chemical exposure was maintained throughout the 92 ± 2 hr assay with periodic renewal of media (and additives) at 4, 20, 28, 42, and 66 hr of incubation. Inclusion of bioactivation increased the toxicity of CP by two orders of magnitude. The minimal affective concentration in the adult and developmental portions of the assay was decreased from 4000 to 20 µg CP/ml and from 1000 to 4 µg CP/ml, respectively. By limiting the inherent toxicity of the MFO package, it was possible to avoid pulse-type exposures and ensure that all ontogenic stages were exposed to active metabolites. The addition of metabolic activation capacity to an in vitro assay, while not essential, markedly enhances its utility and breadth of application in developmental toxicity safety evaluations.


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