Correlation of 2,3,7,8-Tetrachlorodibenzo- p -dioxin-Induced Apoptotic Cell Death in the Embryonic Vasculature with Embryotoxicity Susannah M. Cantrell,* , ² Jennifer Joy-Schlezinger,‡ John J. Stegeman,‡ Donald E. Tillitt,² and Mark Hannink* ,1 *Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65212; ² Midwest Science Center, National Biological Service, Department of the Interior, Columbia, Missouri, 65201; and ‡Department of Biology, Woods Hole Oceanographic Institute, Woods Hole, Massachusetts 02543 Received April 14, 1997; accepted September 15, 1997 Correlation of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Apop- totic Cell Death in the Embryonic Vasculature with Embryotoxicity. Cantrell, S. M., Joy-Schlezinger, J., Stegeman, J. J., Tillitt, D. E., and Hannink, M. (1998). Toxicol. Appl. Pharmacol. 148, 24 –34. Vertebrate embryos are particularly sensitive to 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for under- standing the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apop- tosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis in- dicated that TCDD-induced programmed cell death in the embry- onic medial yolk vein was mechanistically linked to embryo mor- tality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic develop- ment. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A ex- pression and TCDD-induced programmed cell death. TCDD- induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post- hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds. © 1998 Academic Press TCDD is the prototypic member of a group of widespread environmental contaminants generally described as planar ha- logenated hydrocarbons (PHHs). Most of the toxic effects of TCDD are mediated by a cytosolic receptor complex, the Aryl hydrocarbon Receptor (AhR) (Fernandez-Salguero et al., 1996). The presence of PHHs in the environment is of concern, as PHHs are toxic to a wide range of vertebrate species (Lindstrom et al., 1995; Peterson et al., 1993; Olson et al., 1990; Kleeman et al., 1988; McConnell, 1980). Although there are species differences in the degree of susceptibility to TCDD exposure, the embryonic stage of development is the most sensitive life stage for most species to the toxic effects of TCDD (Zabel et al., 1995; Peterson et al., 1993; Spitsbergen et al., 1991; Wisk and Cooper, 1990). Vascular damage is the most pronounced adverse effect of TCDD exposure during embryonic development of fish and avian species (Peterson et al., 1993). Persistent vascular hem- orrhaging and pericardial edema are key morphologic indica- tors that vascular function is compromised in the developing embryo (Henry et al., 1997; Guiney et al., 1997; Spitsbergen et al., 1991; Wisk and Cooper, 1990). In fishes, TCDD-induced vascular lesions increase in severity as the embryo develops, ultimately resulting in decreased blood flow and concurrent regression of the vascular tissue (Henry et al., 1997; Guiney et al., 1997). Supporting the contention that vascular damage is a key physiological mediator of the embryo toxicity of TCDD, vascular damage, as assessed by TCDD-induced apoptotic cell death in the medial yolk vein, has been demonstrated in medaka embryos prior to the appearance of visible vascular lesions (Cantrell et al., 1996). Taken together, these data sug- gest the hypothesis that TCDD-induced vascular damage is causally linked to the embryo toxicity of TCDD. In the present study we further address this hypothesis by establishing dose- response curves for TCDD-induced apoptotic cell death in the medial yolk vein of TCDD-exposed medaka embryos. Although embryo mortality is often the most sensitive of the observable adverse effects of TCDD exposure in fish, sublethal effects are almost certain to occur. For example, rainbow trout eggs exposed to graded doses of a complex mixture of PHHs developed yolk sac edema and subcutaneous hemorrhaging in the surviving organisms (Walker and Peterson, 1994; Walker et al., 1992; Spitsbergen et al., 1991; Walker et al., 1991). 1 To whom correspondence should be addressed. Fax: (573) 884-4597; E-mail: bcmarkh@muccmail.missouri.edu. TOXICOLOGY AND APPLIED PHARMACOLOGY 148, 24 –34 (1998) ARTICLE NO. TO978309 24 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.