The relevance of covalent binding to mouse liver DNA to the carcinogenic action of hexachlorocyclohexane isomers

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Abstract
[ 3 H]Hexachlorocyclohexane (HCH) was synthesized by chlorination of [ 3 H]benzene prepared by catalytic tritiation of benzene with tritiated water. The isomers of HCH were separated by adsorption chromatography on silica gel. In order to determine the covalent binding to DNA, [ 3 H]HCL was administered to male mice by oral gavage, and liver DNA was isolated via chromatin. The specific radioactivity of the DNA was normalized by the dose administered and expressed in the molar units of the Covalent binding index, CBI = DNA damage/dose = ( μ mol bound HCH/mol DNA nucleotide)/(mmol HCH administered/kg body weight). CBI values of ∼0.2 were found 10 h after the administration of alpha- and gamma-HCH. Enzymatic digestion of the DNA to the nucleosides and h.p.l.c. analysis revealed that ∼40% of the radioactivity co-migrated with the natural nucleosides. At elution volumes known to contain the more lipophilic carcinogen-nucleoside adducts, ∼10% of the radioactivity could be detected. The remaining 50% of the radioactivity eluted with the front, representing a mixture of oligonucleotide-HCH adducts and/or hydrophilic degradation products which were strongly but not covalently associated with intact DNA. Therefore, a true CBI of 0.02–0.1 must be expected both for alpha- and gamma-HCH. This CBI is by a factor of 10 5 –10 6 below the value found with the strongest DNA-binding carcinogens like aflatoxin B 1 or dimethylnitrosamine and is unlikely to be decisive for the liver tumor induction in mice because of the following additional findings: (i) Both isomers gave rise to similar levels of DNA damage although the alpha-isomer is a much more potent tumor inducer. This similarity was seen not only at the time of maximum binding but up to 10 days after oral administration; (ii) three mouse strains with apparently different susceptibility to tumor induction by gamma-HCH could not be distinguished with respect to DNA binding; (iii) the level of DNA binding of alpha-HCH (CBI = 0.02–0.1) is more than three orders of magnitude lower than would be expected if the mechanism of tumor induction was by genotoxicity mediated by DNA-binding. For a preliminary investigation on a potential stimulatory effect on liver DNA replication and cell division, [ 14 C]thymidine was administered i.p. 3.5 h before sacrifice of the [ 3 H]HCH-trated mice. The alpha-isomer was found to be more potent than the gamma-isomer in this respect. Taken together, our data allow the conclusion that the non-mutational processes must be more important for the carcinogenicity of HCH.