Toxicological Sciences 53, 118-121 (2000)
Copyright © 2000 by the Society of Toxicology
Comparative Pathogenesis of Haloacetic Acid and Protein Kinase Inhibitor Embryotoxicity in Mouse Whole Embryo Culture
,1,2
* Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599–7270; and
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Haloacetic acids (HAs) are embryotoxic contaminants commonly found in drinking water. The mechanism of HA embryotoxicity has not been defined, but may be mediated in part by protein kinase C (PKC) inhibition. This study was conducted to evaluate the pathogenesis of HA embryotoxicity, and to compare these data with those from specific (Bis I) and non-specific (staurosporine) inhibitors of PKC. Embryos were incubated for varying times with several HAs, Bis I, staurosporine, or Bis V (a negative control). Cell cycle analysis was performed by flow cytometry following nuclear staining with propidium iodide; apoptosis was evaluated by fluorescence microscopy following LysoTracker staining. At concentrations producing 100% embryotoxicity with no embryolethality, only staurosporine perturbed the cell cycle. However, flow cytometry revealed accumulation of sub-G1 events (an apoptotic indicator) across time with bromochloroacetic acid, dichloroacetic acid, and staurosporine, but not dibromoacetic acid, Bis I, or Bis V. Sub-G1 events were particularly prominent in the head region, and remained at control levels in the heart. LysoTracker staining confirmed a similar pattern of apoptosis in the intact embryo; BCA and DCA produced intense staining in the prosencephalon, with virtually no staining in the heart. These data indicate that while cell-cycle perturbation may not mediate the pathogenesis of HA embryotoxicity, these agents do induce embryonic apoptosis. In addition, the lack of Bis I-induced apoptosis indicates that PKC inhibition is unlikely to be the sole mediator of HA embryotoxicity.
Key Words: disinfection byproducts; haloacetic acids; embryotoxicity; whole-embryo culture; protein kinase C; apoptosis; cell cycle.
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