This research will test the hypothesis that substitution of foreign metal ions for Zn2+ in finger-loop domains of certain gene-regulating proteins constitutes a molecular mechanism for metal-induced genotoxicity and teratogenicity. The experiments will be performed upon the frog, Xenopus laevis, and will be focused on two Zn-finger proteins (TFIIIA, Sp1) and four transition metal ions (Ni2+, Co2+, Cu2+, Cd2+). Metals will be analyzed by atomic absorption spectrometry and by liquid scintillation counting of 63Ni and 109Cd. Embryotoxicity and teratogenicity will be determined by morphological and molecular assays. The 1st aim is to administer NiCl2, CoCl2, CuCl2, and CdCl2 to juvenile Xenopus females in order to achieve partial substitutions of the metals for Zn2+ in TFIIIA of previtellogenic oocytes. TFIIIA, isolated from oocytes of control and metal-treated females, will be analyzed for metal content and absorption spectra and tested in vitro for DNA-binding, transcriptional activation, and induction of DNA damage by generating oxygen free radicals. The 2nd aim is to substitute graded amounts of Ni2+, Co2+, Cu2+, and Cd2+ in vitro for Zn2+ in TFIIIA, to determine if the proportions of foreign metals correlate with functional alterations of TFIIIA, tested by the methods cited above. The 3rd and 4th aims are to determine the toxicity and teratogenicity of Ni2+, Co2+, Cu2+, and Cd2+ in embryos exposed to the metals for 96 h, beginning at the blastula stage, using the standard FETAX assay, and to monitor the embryos through metamorphosis to assess visceral, skeletal, and other anomalies. The 5th aim is to detect possible teratogenic effects of metals early in embryogenesis, by exposing embryos to the metals for 3 h after first cleavage, and determining the interval to the onset of transcription of the cytoskeletal actin gene. Since correct activation of the actin gene, which normally occurs at the mid-gastrula stage, is crucial for embryonic development, actin gene activation should provide an endpoint to assay for teratogens that act prior to tissue differentiation, when morphological endpoints are unsatisfactory. If metal exposures inhibit or retard actin gene activation, the metals will be substituted in vitro for Zn2+ in purified Sp 1, to see if the metal substitutions impair Sp 1 binding to a GC-box element that is upstream from the actin gene initiation site, thus elucidating a possible mechanism for teratogenesis. This research will (a) provide valuable data regarding the embryotoxicity and teratogenicity of divalent Ni, Co, Cu, and Cd, reducing uncertainty of reproductive risk assessments for these metals, (b) test a mechanistic hypothesis for metal-induced genotoxicity, (c) provide a promising new assay for teratogens that act early in embryogenesis, and (d) reduce rodent utilization in toxicological research, by demonstrating the feasibility of conducting molecular studies of metal genotoxicity in an amphibian species.
