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Four halobenzoquinones (HBQs), 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), and 2,6-dibromobenzoquinone (DBBQ), have been recently confirmed as disinfection byproducts (DBPs) in drinking water; however, their toxicological information is scarce. Here, we report that HBQs are cytotoxic to T24 bladder cancer cells and that the IC50 values are 95 μM for DCBQ, 110 μM for DCMBQ, 151 μM for TCBQ, and 142 μM for DBBQ, after a 24-h exposure. The antioxidant N-acetyl-l-cysteine (NAC) significantly reduces the cytotoxicity induced by the four HBQs, supporting the hypothesis that oxidative stress contributes to the cytotoxicity of HBQs. To further explore the oxidative mechanisms of cytotoxicity, we examined HBQ-induced production of reactive oxygen species (ROS) in T24 cells, and measured 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyls, and malondialdehyde (MDA) adducts of proteins, markers of oxidative damage to DNA, proteins, and lipids, respectively. All four HBQs generated intracellular ROS in T24 cells in a concentration-dependent manner. HBQs also produced 8-OHdG in genomic DNA of T24 cells, with the highest levels of 8-OHdG induced by DCMBQ. Protein carbonylation was significantly increased in T24 cells that were incubated with each of the four HBQs for 24 h. However, MDA adduct formation, a marker of lipid peroxidation, was not affected by any of the four HBQs tested. These results suggest that the ROS-induced oxidative damage to DNA and protein carbonylation are involved in the observed toxicity of HBQs in T24 cells.

Ovotransferrin is a glycoprotein well-known for its iron-binding property. Ovotransferrin was reported to have antioxidative properties, but the presence of antioxidant peptides within the protein has not been reported. The purpose of the study was to characterize the antioxidant peptides within ovotransferrin. Ovotransferrin was sonicated and hydrolyzed by thermolysin, and peptides from the hydrolysate were fractionated by ion-exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Fourteen peptides derived from ovotransferrin were characterized using LC-MS/MS, and their oxygen radical absorbance capacity (ORAC) values were determined using synthetic peptides. Two tetrapeptides (Trp-Asn-Ile-Pro and Gly-Trp-Asn-Ile) showed the highest antioxidant activity. Interestingly, the addition of amino acid residues to either the N or C terminus of the two peptides decreased the antioxidant activity, suggesting that the motif of Trp-Asn-Ile is responsible for the high antioxidant activity.

Inhibition of DNA repair processes has been suggested as one predominant mechanism in arsenic co-genotoxicity. However, the underlying mode of action responsible for DNA repair inhibition by arsenic remains elusive. To further elucidate the mechanism of repair inhibition by arsenic, we examined the effect of trivalent inorganic and methylated arsenic metabolites on the repair of benzo(a)pyrene diol epoxide (BPDE)-DNA adducts in normal human primary fibroblasts and their effect on repair-related protein expression. We observed that monomethylarsonous acid (MMA(III)) was the most potent inhibitor of the DNA repair. MMA(III) did not change the expression levels of some key repair proteins involved upstream of the dual incision in the global nucleotide excision repair (NER) pathway, including p48, XPC, xeroderma pigmentosum complementation group A (XPA), and p62-TFIIH. However, it led to a marked impairment of p53 induction in response to BPDE treatment. The abrogated p53 expression translated into reduced p53 DNA-binding activity, suggesting a possibility of downregulating downstream repair genes by p53. A p53-null cell line failed to exhibit the inhibitory effect of MMA(III) on NER, implicating a role for p53 in the NER inhibition by MMA(III). Further investigation revealed that MMA(III) dramatically inhibited p53 phosphorylation at serine 15, implying that MMA(III) destabilized p53 by inhibiting its phosphorylation. Because p53 is required for proficient global NER, our data suggest that arsenic inhibits NER through suppressing p53 induction in response to DNA damage in cells with normal p53 gene expression.

Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding.