DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol.
The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.
Nagai
F
F
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan.
Ushiyama
K
K
Satoh
K
K
Kano
I
I
D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage; D004262:DNA Restriction Enzymes; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects*; D016166:Free Radical Scavengers; D005659:Fungicides, Industrial / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats
D016227:Benzoquinones; D001713:Biphenyl Compounds; D003574:Cytochrome c Group; D004278:DNA, Superhelical; D016166:Free Radical Scavengers; D005659:Fungicides, Industrial; D006873:Hydroquinones; D009243:NAD; C029322:phenylbenzoquinone; D009249:NADP; C004369:2-phenylphenol; D004262:DNA Restriction Enzymes; C066330:phenylhydroquinone