Exposure–Disease Continuum for 2-Chloro-2- Deoxyadenosine, a Prototype Ocular Teratogen. 1. Dose-Response Analysis JUDITH A. WUBAH, 1 R. WOODROW SETZER, 2 CHRISTOPHER LAU, 2 JEFFREY H. CHARLAP, 1 AND THOMAS B. KNUDSEN 1 * 1 Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 2 National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 ABSTRACT Background: Treatment of pregnant mice with 2-chloro- 2'-deoxyadenosine (2CdA) on day 8 of gestation induces microphthalmia through a mechanism coupled to the p53 tumor suppressor gene. The present study defines 2CdA dosimetry with respect to exposure (pharmacokinetics), p53 protein induction, and disease (microphthalmia). Methods: Pregnant CD-1 mice dosed with 0.5–10.0 mg/kg 2CdA on day 8 provided fetuses for teratological evaluation; 2CdA was measured by HPLC in the antime- sometrium through 180 min postexposure, and p53 was assessed with immunostaining of the embryo through 270 min. 5'-/3'-RACE was used to sequence the candi- date gene for 2CdA bioactivation from target cells. Results: Microphthalmia appeared first in the dose-re- sponse curve. The highest 2CdA dose having no observ- able adverse effect (NOAEL) was 1.5 mg/kg; the benchmark dose that produced an extra 5% risk of microphthalmia (BMD 5 ) was 2.5 mg/kg, and the lower confidence limit (BMDL) was 2.0 mg/kg. Pharmacokinetic parameters for doses encompassing the threshold (1.5– 2.5 mg/kg) were modeled at 1.0 –1.8 M (C max ) and 30 – 80 M-min (AUC). The p53 response was not de- tected below the BMDL; however, a low-grade response appeared 4.5 hr after a teratogenic dose (5.0 mg/kg), and high-grade induction followed an embryolethal dose (10.0 mg/kg). RACE identified a novel splice variant of mitochondrial deoxyguanosine kinase, dGK-3, as the likely candidate for 2CdA bioactivation in the embryo. Conclusions: Microphthalmia represented the critical effect malformation of 2CdA. The findings suggest a mitochondrial mechanism for 2CdA bioactivation, lead- ing to an embryonic p53 response only after 2CdA elimination and implying pharmacodynamic coupling to the exposure– disease continuum. Teratology 64:154 –169, 2001. Published 2001 Wiley-Liss, Inc. † INTRODUCTION Biologically based dose-response (BBDR) models rep- resent a new stage in the evolution of risk assessment for developmental toxicity (Kavlock and Setzer, ’96). These models integrate the pharmacokinetic and phar- macodynamic properties of a chemical with the under- lying biomolecular changes leading to disease states in the embryo (Gaylor and Razzaghi, ’92; O’Flaherty and Clarke, ’94; Shuey et al., ’94). BBDR models are itera- tive, incorporating new information that becomes available in cell and developmental biology. Conse- quently, BBDR models may improve the scientific basis of risk assessment in several possible ways, including (1) formulation of testable hypotheses pertaining to critical modes of action of developmental toxicants; (2) building the framework for serial translation of empir- ical dose-response relationships into a mechanistic model for toxicant-induced perturbations leading to dysmorphogenesis; (3) predicting interspecies homol- ogy of responses to environmental chemicals; (4) reduc- ing uncertainties in the shape of the dose-response curve for exposure levels below experimental observa- tion; (5) providing risk prediction in different model systems; and (6) expressing, in biomathematical terms, the quantitative estimation of human risk (Lau et al., ’00). To model complex processes in developmental toxic- ity, it makes sense to work with prototype agents that invoke fundamental disease pathways in the develop- ing embryo. One candidate is 2-chloro-2'-deoxyade- nosine (2CdA). This purine nucleoside analogue is cy- totoxic by virtue of its similarity to 2'-deoxyadenosine, a metabolic toxin that invokes deoxyadenylate (dATP) stress imbalance within specific cell types (Cohen et al., Grant sponsor: National Institute of Environmental Health Sciences; Grant number: T32 ES07282; Grant sponsor: NRSA; Grant sponsor: National Institute of Child Health and Human Development; Grant number: F31 HD08167; Grant sponsor: Environmental Protection Agency; Grant number: EPA-CR 824 445-01. *Correspondence to: Thomas B. Knudsen, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107. E-mail: thomas.knudsen@mail.tju.edu Received 28 August 2000; Accepted 7 May 2001 TERATOLOGY 64:154 –169 (2001) Published 2001 WILEY-LISS, INC. † This article is a US government work and, as such, is in the public domain in the United States of America.