Table 1. A list of studies on possible involvement of ROS including the hydroxyl radical in mutagenicity Author de Kok 1992 Akman 1992 Dizdaroglu 1993 Brennan 1994 Yoshida 1998 Chlopkiewicz 1993 2Chloropyridine Mutagen Fecapentaene12 Doxorubicin Phorbol12acetate 13myristate (PMA) Hydrogen peroxide pAminophenol Edenharder and Grunhage 2003 tButyl hydroperoxide (BHP), Cumene hydroperoxide (CHP) Grey 2003 Hydrogen peroxide idea that the hydroxyl radical is a putative cause of Cr(VI)-induced carcinogenesis. Epidemiological studies have indicated that people exposed to high levels of arsenic are prone to skin, bladder, liver, and lung cancers,(31) and the skin is one target for arsenic toxicity such as keratinocytic tumors including basal cell carcinoma and squamous cell carcinoma, since it was reported that organic and inorganic arsenic can be absorbed through the skin in animals and humans.(32) Regarding the underlying mechanism by which arsenic induces skin cancer, Shi et al.(33) examined the chemical nature of specific ROS, studied the interrelationship among these species, and identified the specific species responsible for the subsequent DNA damage in a spontaneously immortalized keratinocyte cell line. They detected the formation of superoxide anion and hydrogen peroxide in keratinocytes incubated with arsenite(III) concomitant with DNA damage. Catalase eliminated hydrogen peroxide and reduced the arsenite(III)-mediated DNA damage. In contrast, SOD significantly increased the arsenite(III)-mediated damage possibly by enhancing the production of hydrogen per- oxide and hydroxyl radicals. Since sodium formate, a competitive scavenger for the hydroxyl radical, and deferoxamine, a metal chelator, both reduced the DNA damage, exposure to arsenite(III) may generate superoxide anions and hydrogen peroxide, and hydroxyl radicals derived from hydrogen peroxide, may play an important role in arsenic-induced skin carcinogenicity. However, unlike Cr and arsenic, the involvement of hydroxyl radicals in vanadium-induced carcinogenesis was reported to be minimal.(34) A correlation between exposure to airborne vanadium particles and the incidence of cancer in residents of metropolitan areas has been indicated by epidemiological studies.(35,36) It was reported that mutations and DNA-protein crosslinks were induced by vanadate(V) in cultured mammalian cells,(37) and the transforming activity of vanadate(V) was found in BALB/3T3 cells,(38) sug- gesting that vanadate(V) may act both as an initiator and as a promoter of morphological transformation in cultured mammalian cells. More recently, Ding et al.(34) reported that the toxicity and carcinogenicity of vanadium arise from elevated levels of ROS leading to activation of the transcription factor activator protein-1 (AP-1). To investigate the possible activity of vanadium in the activation of AP-1, they treated mouse epidermal JB6P1 cells stably transfected with an AP-1 luciferase reporter plasmid with various concentrations of vanadate(V). SOD and catalase inhibited AP-1 activation induced by vanadate(V), indicating the involve- ment of superoxide anions and/or hydrogen peroxide in the mechanism of vanadate(V)-induced AP-1 activation. However, T. Kanno et al. Assay Bacterial reverse mutation DNA base modification DNA base modification Bacterial reverse mutation Chromosomal aberration Bacterial reverse mutation Bacterial reverse mutation Bacterial reverse mutation Brief summary Mutagenicity induced by fecapentaene12 in Salmonella strains was suppressed by scavengers of hydroxyl radical. DNA base modifications were induced in isolated human chromatin by NADH dehydrogenasecatalyzed reduction of doxorubicin, which causes hydroxyl radical production. Exposure of target cells to PMAactivated human PMNs caused DNA base modifications in the cells. Mutagenicity induced by 2chloropyridine in Salmonella strains was suppressed by scavengers of hydroxyl radical. Hydrogen peroxideinduced intrachromosomal and inter chromosomal recombination in Saccharomyces cerevisiae were suppressed by DMSO. Mutagenicity induced by paminophenol in E. coli WP2uvrA/ pKM101 was suppressed by DMSO or catalase. Mutagenicity induced by BHP or CHP in Salmonella typhimurium TA103 was reduced by some flavonols, which possess radical scavenging effects. Mutagenicity induced by hydrogen peroxide in Salmonella typhimurium TA102 was reduced by ascorbic acid and DMSO, both of which are effective scavengers of hydroxyl radical. sodium formate failed to inhibit vanadate(V)-induced AP-1 activation, suggesting that vanadium, like other classes of tumor promoters, transactivates AP-1-dependent gene expression which might be attributable to the generation of superoxide anions and hydrogen peroxide, but not hydroxyl radicals. Beside the involvement of ROS in the carcinogenicity of heavy metals, Elliot et al.(39) reported that carcinogenicity observed in rodents with certain peroxisome proliferators might be linked with ROS including the hydroxyl radical. In their study, ESR using the spin trap DMPO was employed to measure hydroxyl radical production in liver peroxisome-enriched fractions isolated from male Alpk/Ap rats administered chemicals such as di(2- ethylhexyl)phthalate, clofibrate and methyl clofenapate, all of which are known to cause peroxisome proliferation. The results showed that the rate of production of hydroxyl radicals was greater in fractions from the treated animals, raising a hypothesis linking such ROS to carcinogenicity observed in rodents treated with certain peroxisome proliferators. Overview of the Hydroxyl Radical and Carcinogenicity Table 2 summarizes studies on the involvement of ROS in- cluding the hydroxyl radical in chemically-induced carcinogenicity. Only one study, where the possible involvement of ROS in vanadium-induced carcinogenicity was examined, expressed skepticism about the involvement of the hydroxyl radical.(34) All the other studies proposed the hydroxyl radical to be involved in chemically-induced carcinogenicity. Notably, the carcinogenicity of heavy metals such as Cr and arsenic may involve hydroxyl radicals produced by Fenton-like reactions.(28–30) Conclusion Despite a lack of direct evidence in vitro, the hydroxyl radical is likely a causative factor for bacterial reverse mutations and DNA base modifications in yeast and mammalian cells induced by mutagens, because the scavenging of hydroxyl radicals resulted in the cancellation or attenuation of these mutagenic responses. Similarly, the hydroxyl radical possibly plays a role in heavy metal-induced carcinogenicity since these metals can mediate Fenton-like reactions with hydrogen peroxide, resulting in hydroxyl radicals as illustrated in Fig. 2. The generation of superoxide anion following the exposure of cells to heavy metals such as Cr and arsenic gives rise to hydrogen peroxide via a dismutation reaction J. Clin. Biochem. Nutr. | July 2012 | vol. 51 | no. 1 | 11 ©2012 JCBN