Superfund Research Program

Molecular Mechanisms of Complex Mixture Toxicity

Project Leader: Alvaro Puga
Grant Number: P42ES004908
Funding Period: 2001 - 2006

Project-Specific Links

Final Progress Reports

Year:   2005 

Research, led by UC Environmental Health investigators Timothy Dalton, Alvaro Puga, and Ying Xia, has attempted to elucidate the mechanisms of arsenic cytotoxicity.  A report, published by the team in the July 2005 issue of Toxicological Sciences addresses the potential mechanisms of arsenic toxicity.  Results in this study, using the most hazardous environmental form of arsenic, confirmed that the primary cellular effect of exposure is a significant increase in the level of reactive oxygen species within the cell.  Under normal physiological conditions, the molecule glutathione scavenges the reactive oxygen species, maintaining redox equilibrium within the cell.  This depletion of glutathione acts as a signal triggering de novo synthesis.  Unmediated reactive oxygen leads to harmful catabolic reactions, altering cell signaling, eventually causing DNA damage, and apoptotic cell death.  Arsenic dose response assays were performed with cells unable to produce normal levels of glutathione.  These cells were significantly more sensitive to arsenic than wild-type controls.  Pretreatment with a potent antioxidant significantly inhibited apoptosis but did not restore glutathione levels to normal levels, suggesting that cellular oxidation state determines sensitivity to arsenic but does not determine survival capability.  Rather, the ability of the cell to continue to synthesize glutathione controls the outcome of arsenic exposure.

 

In addition to generating reactive oxygen within the cell, arsenic has been reported to bind with high affinity to adjacent sulfhydryl molecules on protein.  Through this binding, arsenic alters the structure of the protein, subsequently inhibiting enzyme function.  While the mechanisms of arsenic toxicity and PAH toxicity have been analyzed in depth, relatively little research has considered the effects of simultaneous exposure to the two substances.  The research team, under the direction of Drs. Puga and Xia, report in the Aug 2005 issue of Molecular Pharmacology that exposure to complex arsenic-PAH mixtures poses a greater hazard than singular exposure to either substance.  Once again addressing cellular and genetic effects of arsenite exposure, the group investigated the impact of arsenic on the well-defined Aryl Hydrocarbon Receptor (AHR) pathway.  The AHR is a cytosolic transcription factor that, when activated, translocates to the nucleus, activating transcription of genes responsible for detoxification.  The AHR has a potent affinity for PAHs, such as dioxin (Tri-Chloro-Dibenzo-p-Dioxin: TCDD), a byproduct of incineration and smelting, and benzo[a]pyrene, produced through incomplete combustion of organic materials.  After activation of the AHR by these ligands, the receptor enters the nucleus as part of a complex that includes the AHR nuclear translocator (ARNT) and Heat Shock Protein (Hsp90) molecules.  Arsenic also induces translocation of the AHR, but no HsP90 was detected in the nuclei of arsenite-treated cells.  The change suggests a possible alternate mechanism for AHR activation.  To test this hypothesis, mouse hepatoma cells were treated with 3M4NF, which blocks ligand-induced AHR translocation, followed by treatment with arsenite or TCDD.  As expected, 3M4NF blocked AHR translocation in TCDD-treated cells, but nuclear AHR was still detected in arsenite-treated cells. 

 

Realizing that they had discovered an alternate mode of AHR activation, the team then compared the level of gene expression resulting from activation through each pathway.  Using the cytochrome P450 gene, Cyp1a1, as a reference, RNA levels were measured after treatments with arsenite, TCDD or both compounds.  Arsenite alone caused a 2-5-fold increase in expression over untreated control levels; TCDD increased Cyp1a1 by 35- to 40-fold, and arsenite with TCDD induced Cyp1a1 60-fold over control.  The separate mechanisms of AHR activation achieved different outcomes of gene expression, confirming the existence of an alternative mode of AHR transactivation.  While the classical, ligand-mediated pathway had a greater effect on gene expression than arsenite alone, exposure to both compounds caused the most significant induction of Cyp1a1.  This report presents compelling evidence that PAHs and arsenic are more hazardous when acting as comutagens.