genesis. Although no specific conditions that resulted in the direct formation of oxygen radicals from FP-12 could be defined, the formation of hydroxyl radical spin adducts was demonstrated after peroxidation of FP-12 in incubations with the spin-trapping agent 2,2,6,6-tetramethyl-piperidine. Furthermore, in mutagenicity assays to examine the effect of removing hydroxyl radicals with respect to the genotoxic potential of FP-12, the scavenging reduced the number of revertants in Salmonella strains TA100, TA102 and TA104, suggesting a partial involvement of the hydroxyl radical in the mutagenic effect with the formation of superoxide anions after the peroxidation of FP-12, which is subsequently converted to hydroxyl radicals by the iron-catalyzed Haber-Weiss reaction. Chlopkiewicz et al.(10) examined whether the hydroxyl radical contributes to the mutagenicity of 2-chloropyridine in Salmonella typhimurium. They found that the mutagenicity was effectively suppressed by scavengers of the hydroxyl radical and was not inhibited by the addition of superoxide dismutase (SOD) or catalase. However, the results did not clarify whether the mutagenicity of 2-chloropyridine was caused by hydroxyl radicals or whether other oxygen species generated intracellularly were in- volved, because SOD or catalase added to the medium could not scavenge the superoxide anion or H2O2 generated within the cell. In a study by Edenharder and Grunhage, the mutagenicity caused by t-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102 was effectively reduced by some flavonols.(11) However, although they concluded that in the Salmonella/ reversion assay with strain TA102, antimutagenic activities of flavonoids against the peroxide mutagens t-butyl hydroperoxide and cumene hydroperoxide are mainly caused by radical- scavenging effects, it was not clarified which radical species were involved. Similarly, hydrogen peroxide-induced mutagenicity in Salmonella typhimurium TA102 was assumed to be attributable to hydroxyl radicals.(12) In the study, it was proposed that the hydrogen peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA, because protection against the mutagenicity was achieved with ascorbic acid and dimethyl sulfoxide (DMSO), both effective scavengers of hydroxyl radicals. Yoshida et al.(13) reported the mutagenicity of p-aminophenol (p-AP) in Escherichia coli WP2uvrA/pKM101 and its relevance to oxidative DNA damage. Since the mutagenic activity of this compound was suppressed with the addition of DMSO or catalase, they suggested the involvement of active oxygen species in the mutagenic process induced by p-AP. Thus, they speculated hydroxyl radicals derived from the reaction between hydrogen peroxide and Fe(III) (Fenton reaction) to be acting in Escherichia coli WP2uvrA/pKM101 during the mutation by p-AP. Beside the mutagenicity in bacteria, oxidative DNA damage and chromosomal aberrations have been reported in yeast and mammalian cells. Akman et al.(14) reported DNA base modifica- tions induced in isolated human chromatin by the NADH dehydro- genase-catalyzed reduction of doxorubicin, an antineoplastic benzanthroquinone. Since the reduction of doxorubicin caused hydroxyl radical production, it was suggested that the flavoenzyme- catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, which might be a possible mechanism for the muta- genicity of doxorubicin in vivo. Dizdaroglu et al.(15) found that phorbol-12-acetate-13-myristate (PMA)-activated human poly- morphonuclear leukocytes (PMNs) caused base modifications in target cell DNA in vivo, suggesting that potentially promutagenic bases contribute to the mutagenicity of activated PMNs via the generation of reactive oxygen species including the hydroxyl radial. With regard to chromosomal aberrations, it was reported that of five oxidative mutagens, methyl viologen (paraquat), mito- mycin C, phenylhydrazine, cumene hydroperoxide and hydrogen peroxide, only hydrogen peroxide increased the frequency of both intrachromosomal recombination and interchromosomal recombi- 10 nation in the yeast Saccharomyces cerevisiae.(16) A role for the hydroxyl radical in the effect of hydrogen peroxide on recombina- tion was indicated by the ability of the radical scavenger DMSO to significantly inhibit the induction of both intrachromosomal and interchromosomal recombination by hydrogen peroxide. Overview of the Hydroxyl Radical and Mutagenicity As described above, there have been many reports on the involvement of hydroxyl radicals in mutagenicity in bacteria, yeast and mammalian cells. Table 1 summarizes these studies. Of these, only one tried to detect reactive oxygen species including the hydroxyl radical by using ESR spin trapping to elucidate the underlying mechanism of FP-12-induced mutagenicity in Salmonella.(9) The rest of the studies examined indirectly the kinds of ROS involved in chemically-induced mutagenicity or DNA base modifications in bacteria, yeast or human cells by using ROS scavengers such as DMSO for the hydroxyl radical and catalase for hydrogen peroxide. These findings suggest the possible involvement of hydroxyl radical in chemically-induced muta- genicity in vitro. Hydroxyl Radical and Carcinogenicity The possible involvement of hydroxyl radicals in chemically induced carcinogenicity has been reported in several papers. The hydroxyl radicals produced by Fenton-like reactions may be involved in the carcinogenicity of iron (Fe), copper (Cu) and other heavy metals such as chromium (Cr) and arsenic. Fe and Cu are essential elements for the functions of various enzymes and cellular proteins, and are regulated by certain biomolecules such as transferrin receptor for Fe and Cu transporter-1 for Cu.(17,18) It was reported that accumulation of Fe is associated with colorectal cancers in humans, and a genetic hemochromatos is associated with increased risk of human hepatocellular tumors.(19) Liver tumor promotion was also evident after Fe overloading in a rat two-stage hepatocarcinogenesis study.(20,21) It was also proposed that lipid peroxidation initiated by Fe-mediated oxidative stress possibly including hydroxyl radicals is associated with renal carcinogenesis.(22,23) With regard to Cu, it was reported that increased Cu storage due to impaired cellular Cu transporters can cause serious liver diseases.(24) Especially, liver cell injury caused by generating ROS including hydroxyl radicals through excess Cu accumulation can cause tumor promotion, possibly by inducing oxidative DNA damage.(25–27) Ye et al.(28) reported that Cr(VI) in combination with glutathione reductase enhanced the activity of nuclear factor (NF)-κB in Jurkat cells. This activation of NF-κB was decreased by a metal chelator, diethylenetriaminepentaacetic acid or catalase, but increased by SOD. ESR measurements using 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) confirmed that the incubation of Cr(VI) with Jurkat cells in the presence of glutathione reductase generated hydroxyl radicals. In addition, the hydrogen peroxide enhanced hydroxyl radical generation and also enhanced Cr(V) formation, suggesting Cr(VI)-mediated NF-κB activation to be involved in the mechanism of Cr(VI)-induced carcinogenicity. According to Liu et al.,(29) the Cr(VI)-glutathione complex is able to generate hydroxyl radicals in the presence of molecular oxygen in aqueous medium. Since a catalase inhibited the production of hydroxyl rad- icals while hydrogen peroxide enhanced it, the hydroxyl radical was generated via a Fenton-like reaction. The results imply the involvement of the Cr(IV)-mediated production of the hydroxyl radical in the mechanism of Cr(VI)-induced carcinogenesis. More recently, Hojo et al.(31) successfully detected the hydroxyl radical in the blood of mice after acute Cr(VI) intake by applying ESR spin-trapping. The ESR spectrum of the blood of mice given 4.8 mmol Cr(VI)/kg body weight revealed 7.37 μM DMPO-OH, a spin adduct of DMPO and the hydroxyl radical, supporting the doi: 10.3164/jcbn.11105 ©2012 JCBN