Epidemiological evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be associated with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, especially for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degradation [Bal et al. (1997) Chem. Res. Toxicol., 10, 906-914 and 915-921]. In the present study, effects of HP21-15 on Ni(II)- and Cu(II)-mediated DNA oxidation by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and production of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21-15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21-15. The effect of HP21-15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21-15 strongly suppressed the oxidative activity of Cu(II)/H2O2 in regard to all bases in DNA. Consistently with the above, the electron spin resonance/spin trap measurements revealed greater and more persistent generation of OH and O2-like oxidants from H2O2 by the Ni(II)-HP21-15 complex than by the Cu(II)-HP21-15 complex (no O2 was detected). Both complexes were also found to bind to DNA more strongly than HP21-15 alone. The results indicate that protamine P2 is capable of binding Ni(II) and Cu(II) and, in this way, attenuating the mediation of oxidative DNA damage by Cu(II), but not Ni(II). The effects found may be mechanistically involved in the reproductive toxicity and carcinogenicity of metals.
Laboratory of Comparative Carcinogenesis, National Cancer Institute, FCRDC, Frederick, MD 21702