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Final Progress Reports: University of Cincinnati: Mechanisms of Mutagenesis of Metals and PAH/Metal Mixtures

Superfund Research Program

Mechanisms of Mutagenesis of Metals and PAH/Metal Mixtures

Project Leader: Kathleen Dixon (University of Arizona)
Grant Number: P42ES004908
Funding Period: 2001 - 2006

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Final Progress Reports

Year:   2005 

Arsenic is a human carcinogen that is widespread in the environment and present in many of the nation’s Superfund hazardous waste sites.  Often, arsenic is found in complex mixtures in combination with other carcinogenic agents, particularly polycyclic aromatic hydrocarbons (PAHs).  Experimental evidence suggests that arsenic does not act alone, but enhances the carcinogenic activity of other environmental agents.  Dr. Dixon’s project focuses on understanding this cooperative effect of arsenic with other environmental agents.  Most carcinogenic agents cause DNA damage and genetic alterations (mutations) as early steps in the process of carcinogenesis.  While the DNA damaging and mutagenic activity of arsenic alone appears to be weak in most assay systems, arsenic has the ability to enhance the mutagenic activity of other carcinogenic agents.  Such synergy has been hypothesized to involve effects on DNA repair and/or cell cycle checkpoints.


Cell cycle checkpoint pathways are surveillance mechanisms that help maintain genomic integrity. The absence of normal checkpoint functions can lead to premature progression through the cell cycle, insufficient time for DNA repair or failure to eliminate damaged cells. Any of these events will lead to an increased risk of genomic instability and its associated risk of malignant transformation. In order to investigate the effects of arsenic on cell cycle checkpoints, Dr. Dixon’s group studied the ability of arsenic treatment alone to activate/deactivate checkpoints, as well as arsenic’s ability to modulate checkpoints induced by ultraviolet light (UV) irradiation. The study showed that sodium arsenite alone at 5 µM did not markedly alter cell cycle progression in HeLa cells other than initiating an M-phase arrest. In contrast, following UV irradiation arsenite restored the G1 checkpoint and enhanced the S and G2 checkpoints in HeLa cells. These results suggest that arsenic does not inhibit the activation of DNA damage checkpoints after UV, and its function as a co-mutagen or co-genotoxin most likely does not occur via cell cycle checkpoint suppression. Instead, the observed enhancement of cell cycle checkpoints suggests an increase in DNA damage signaling, perhaps due to an inhibition of DNA repair.


Dr. Dixon’s observations that arsenic interferes with DNA repair led to the investigation of phosphorylation of Replication Protein A (RPA). RPA is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. The researchers demonstrated that arsenic enhances and prolongs the DNA damage-induced hyperphosphorylation of RPA, consistent with inhibition of DNA repair. They have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. Dr. Dixon’s group has mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, they confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

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