# Presentation: 897668

## Defining the Role of Biomethylation and Reactive Oxygen Species in Arsenic Carcinogenesis  

**Defining the Role of Biomethylation and Reactive Oxygen Species in Arsenic Carcinogenesis****  **
- Stephen Nesnow1, David Demarini1, Susan Hester1, Kirk Kitchin1, Andrew Kligerman1, Leon King1, Jeffrey Ross1,
- Sheau-Fung Thai1, Witold Winnik1, Douglas Wolf1; Reeder Sams2; Ron Mason3 and Maria Kadiiska3; William Cullen4
-  U.S. EPA; 1ORD, NHEERL; 2ORD, NCEA; 3NIEHS; 4University of British Columbia.

- Exposure to arsenic is worldwide due to both natural and an-made processes (refining, pesticides). Naturally-occurring inorganic arsenic is found in drinking water and several pesticides in commerce are methylated arsenicals. Arsenic-associated human diseases include bladder, lung, skin, and liver cancer. At present, the mode(s) of carcinogenic action of arsenicals in humans is unknown. This data gap prevents a scientifically-defensible quantitative human cancer risk assessment from being established for arsenic. Inorganic arsenic is biotransformed through a metabolic cascade to trivalent methylated forms, some which are highly toxic and genotoxic. Some inorganic and methylated arsenicals are carcinogenic in rodents, but their modes of action are unknown.
- We have evidence that arsenicals induce reactive oxygen species whose formation may explain arsenic’s carcinogenic effects both in rodents and humans.
- We pose the following scientific questions in these mode of action studies addressing human health.
- 1. What is the mode of action of arsenic-induced bladder cancer in rodents and humans?
- 2. What is the role of reactive oxygen species and oxidative stress in arsenic’s toxicity and cancer?
- We will focus our studies on methylated forms of arsenic.

**Research Goals**

**Impact and Outcomes**

**Science Questions**

- One key issue that impinges on the arsenic risk assessment is that of linearity or nonlinearity of the dose-response relationship for tumors. Mode of action studies can help inform which is the more likely response.
- This research is targeted to examine this issue and others and we have already made significant progress toward an understanding of the mode of action of arsenic carcinogenesis.
- Our research on biomethylation of arsenic has already influenced the Office of Water and Office of Pesticide Programs in their ongoing reevaluation of the risk to arsenic exposure.
- This collaborative research effort between EPA, NIEHS, and academia has been influenced in its research direction by the needs of OW and OPP.
- The impact of clarifying the mode of action of arsenic carcinogenesis will be on the establishment of both the arsenic unit cancer risk and the Maximum Contaminant Level (MCL) of arsenic in drinking water and the risk assessments of organoarsenicals.

**Acknowledgments**

- The authors would like to thank all of the staff on the ECD Arsenic Project Team for their assistance.

- Introduction: This broad research effort integrates chemical, biochemical, biological, molecular, and genomic approaches to uncover and delineate the critical events in the induction of bladder cancer in rodents and humans.
- Two key biomethylated arsenicals, the trivalent methylated [DMA(III)] and the pentavalent methylated [DMA(V)], are key intermediates in the arsenic metabolic cascade.  DMA(V) induces rat bladder cancer and DMA(III) has been detected in the urine of DMA(V)-treated rats. We will study important events in the bladder cancer process in rodents and in rodent and human urothelial cell lines: DNA damage (clastogenesis), DNA repair, DNA methylation, cytotoxicity, increased cell proliferation, altered cell cycle control, and apoptosis.
- Our research addresses this problem at the molecular, cellular, and organismal levels. We seek to identify those reactive oxygen species responsible for the critical events involved in the initiation and progression of arsenic carcinogenesis.
- We plan to examine the alterations in gene expression, cell proliferation, and apoptosis in the transitional epithelium of the urinary rat bladder in a series of studies at exposure concentrations at or near some environmental levels.
- We will compare these findings to those obtained from the exposure of rat and human urothelial cells in culture to arsenicals to define the cellular and molecular events associated with arsenic-induced carcinogenicity.

**Conceptual Approach**

**Study Design**

**Results**

**Scientific Conclusions**

**Future Directions**

- The trivalent methylated arsenical DMA(III), trimethylarsine, and dimethylarsine are potent DNA strand breakers.
- DMA(III) is a potent clastogen at micromolar concentrations.
- The ROS form produced by DMA(III) which damages DNA is the hydroxyl radical.
- Morphological changes observed at carcinogenic doses of DMA(V) exposure are indicative of cellular toxicity.
- Categories of genes altered after DMA(V) exposure include apoptosis, cell cycle regulation, proliferation and oxidative stress.
- Molecular changes that may be responsible for DMA(V) induced carcinogenicity are operational even at low doses of 1 and 4 ppm and detectable using gene expression analysis.
- A subset of genes was identified which had increased expression after exposure to carcinogenic doses (40 and 100ppm) as compared to non-carcinogenic doses (1 and 4ppm).
- Our data suggests that changes in gene expression of cancer control pathways is not sufficient to drive the cancer process, but that cellular toxicity present at higher doses is also necessary.

**Identify the biomethylated As species responsible for DNA damage. *****Approaches- examine the DNA damaging potentials of biomethylated As using isolated DNA, in vitro and in vivo. ***
**I****dentify the ROS species responsible for DNA damage and cytotoxicity after DMA(III) exposure.  *****Approaches- use ROS inhibitors, ESR spin traps in isolated and in vivo, histopathology***
**Identify changes in gene expression and molecular pathways in the transitional epithelium of the urinary bladder after exposure to DMA(V) and DMA(III)  Determine if the molecular signatures are dose-dependent.**
**Identify ROS protein damage caused by DMA(V) and DMA(III) in rat bladder transitional epithelial cells for potential uses as human biomarkers.  *****Approaches- measure protein carbonyls, measure oxidized tyrosine-bound proteins***

**Role biomethylation in As Genotoxicity**

- DMA(III) is the most potent arsenical in damaging human lymphocyte DNA by as measured by the comet assay. It also is a spindle cell poison

- In human lymphocytes DMA(III) is the most potent arsenical in inducing chromosomal damage and SCE

**Role of ROS in DMA(III) Genotoxicity**

- DMA(III) induced damage of X174 DNA is reversed by three ROS inhibitors. R = relaxed damaged form;                        S = supercoiled native form

- The predominant ROS form is the hydroxyl radical which was trapped with DMPO and visualized by ESR in incubations of DMA(III) and X174 DNA.

**Working Hypothesis for DMA(V) Rat Bladder Carcinogenesis MOA**
**Initiation-promotion-progression**
- DMA(V) is reduced to DMA(III) and methylated arsines by arsenic reductase in the liver and are excreted into the urine
- DMA(III) and methylated arsines are oxidized in the bladder to the hydroxyl radical and other ROS.
- The hydroxyl radical causes chromosomal rearrangements in the transitional bladder epithelium (TCE). (Genotoxic initiation)
- ROS induce cytotoxicity and cell proliferation the TBE. Cell death and regeneration also occur. (Promotion)
- ROS cause further genomic events leading to chromosomal instability and neoplastic lesions (Progression).

- Light and transmission electron microscopic images of the rat bladder epithelium following exposure to DMA(V) in the drinking water.

- Control

- 100 ppm DMAV

- Control

- 100 ppm DMAV

**Mode of Action Studies in DMA(V)-Induced Rat Bladder Cancer**

**Morphology**
- Light microscopy:  a dose-dependent increase in cytoplasmic vacuolation and hyperchromasia of bladder transitional cells.
- Transmission electron microscopy: vacuolation and decreases in the number of mitochondria
- Scanning electron microscopy:  exfoliation of transitional cells.

**Overview:** Cytotoxicity and increased cell proliferation are associated with a dose dependent increase in tumors that arise from the cells that line the urinary bladder following chronic exposure to DMAV in the diet or drinking water in F344 rats.  Since arsenic-induced neoplasms arise from the transitional epithelium of the urinary bladder, a series of studies have been performed and are planned to investigate multiple endpoints in this cell population including gene expression profiling to understand the molecular events associated with arsenic-induced carcinogenicity following exposure to DMAV.  Female 344 rats exposed to 1, 4, 40 or 100 ppm DMAV in their drinking water for 4 weeks.

**Gene Expression Studies in Rat bladder Epithelium**

- Identification and quantitation of the radical species in the urine of rat bladders after DMA(V) and DMA(III) exposure. *Use ESR-spin trap method, and GC/MS hydroxylated salicylic acid assay. *
- Characterization of DNA damage in rat bladder TBE by DMA(V) and DMA(III). *Measure micronuclei and Comet patterns. *
- Characterization of oxidized proteins in rat bladder TBE by DMA(V) and DMA(III) as a biomarker. *Determine oxidized tyrosines and oxidized proteins.*
- Identify concordant cancer pathways to inform methods for appropriate species extrapolation. *Use gene expression microarray analyses on human and rodent urothelial cell lines after exposure to DMA(V) and DMA(III).*