Superfund Research Program
DNA Adducts as Biomarkers of Exposure and Effect
Risk assessments of National Priority List chemicals are usually based on linear extrapolation of tumor data from high exposures in experimental animals. The 1996 Revised Cancer Risk Assessment Guidelines contain many important changes regarding how future risk assessments will be performed, including the incorporation of more science on the mode of action and dose response of the chemical in question. SBRP-funded researchers at the University of North Carolina at Chapel Hill (UNC), led by Dr. Jim Swenberg, are working to develop a sound scientific understanding of the mode of action and the observed and expected dose response relationship of several of the major hazardous chemicals on National Priority List. Their data will be suitable for use with the revised guidelines and will improve the accuracy of the risk assessments driving site remediation.
The UNC researchers believe that several aliphatic and aromatic chlorinated compounds share a common mode of action - enhanced oxidative stress - which results in indirect damage to DNA and activation of signal transduction pathways involved in gene regulation and cell proliferation. Other members of this group of chemicals cause direct DNA damage. Currently, they are conducting studies to develop biomarkers of exposure and effect to both groups of chemicals. These biomarkers will allow accurate determination of the amount, or molecular dose, that damages DNA and proteins; will enhance examination of the extent and type of both direct genotoxicity and oxidative stress related DNA damage; and will enable risk assessors to more accurately predict risk for cancer.
Vinyl chloride (VC) has been detected at over 100 Superfund sites and is ranked fourth on the ATSDR list of hazardous substances. VC is present in soil, groundwater, aquifers, and wells as the result of inappropriate disposal, accidental spills, and as a product of incomplete biodegradation of chlorinated chloroethenes such as TCE. VC-induced carcinogenicity is believed to occur by genotoxic mechanisms. The enzyme cytochrome P450 oxidizes VC to 2-chlorothylene oxide (CEO), which in turn alkylates to DNA to form 7-(2-oxoethyl)guanine (OEG) and a variety of promutagenic exocyclic base adducts. Although OEG is the major DNA adduct formed by VC, it does not cause mutations. N2,3-ethenoguanine (N2- εG) is formed in relatively small amounts, but has been determined to be both persistent and mutagenic. Scientists in Dr. Swenberg's laboratory have developed a highly specific and sensitive assay for N2-εG (IA/GC/ECNCI/HRMS - immunoaffinity, gas chromatography, electron capture negative chemical ionization, high resolution mass spectrometry) and they are using N2-εG as a biomarker to investigate several aspects of VC mutagenicity.
While it has been demonstrated that VC induces hepatic cancer in humans and rodents, neither experimental nor epidemiologic studies have established a definitive link between VC and brain cancer. By examining the molecular dosimetry of N2-εG in rat brain and liver cells following exposure to VC, Dr. Swenberg's research team found clear evidence of genotoxic attack of hepatic DNA, but determined that N2-εG is not formed in brain due to VC exposure. The same DNA adduct, N2-εG, is present in both liver and brain DNA as a result of endogenous oxidative stress in both control and VC-exposed tissues. While VC does distribute to brain, it is not metabolized to CEO in brain. Likewise, CEO formed in the liver is not transported to the adult rat brain due to its extreme instability. By exposing rats to stable isotope-labeled VC, [13C2]-VC, the researchers were able to measure both the endogenous N2-ε G and the [13C2]- N2-εG in the same animals. These findings indicate that it is unlikely that N2-εG or other VC-induced DNA adducts play a significant role in initiating carcinogenesis in the brain after exposure to VC. Results with weanling rats were less definitive, suggesting age-dependent differences in susceptibility to VC exposure. Additional [13C2]-VC exposures of weanling rats will be needed to clarify these age differences in brain.
In a unique study to investigate the exposure response to a broad range of VC concentrations, the researchers examined the molecular dosimetry of N2-εG in two liver cell types, hepatocytes (HEP) and nonparenchymal cells (NPC). They found that:
- While NPCs are particularly susceptible to VC-induced carcinogenesis, no significant difference in N2-εG concentrations between HEPs and NPCs was seen in any adult exposure group.
- The exposure response curves of HEPs and NPCs were supralinear - with a linear increase from 0 to 100 ppm and a plateau between 100 and 1000 ppm. This is thought to be the consequence of saturation of VC metabolism to CEO.
- Low levels of endogenous N2-εG are found in all tissues examined as a result of lipid peroxidation. Using [13C2]-VC in the exposure study allowed for accurate quantitation of endogenous and VC-induced N2-εG in the same animal. This study showed that the endogenous levels of N2-εG are not affected by exposure to VC.
- The researchers were also able to demonstrate that current occupational exposures of 0.1 to 1.0 ppm VC result in a 5 or 50% increase in N2-εG over that found in non-exposed rats. These data suggest that low ppb exposures to VC found in most Superfund sites could not be distinguished from no exposure.
- N2-εG concentrations were significantly higher in weanling rats than in adult rats, a reflection of greater P450 activity and metabolism of VC to CEO.
- N2-εG is slowly repaired in vivo. The persistence of N2-εG amplifies its mutagenic potential.
These studies strongly support a causal role for N2-εG in VC-induced carcinogenesis and suggest that the molecular dosimetry of N2-εG in liver is predictive of cancer risk. The data do not support VC as a causative factor for the induction of brain tumors. This information also provides mechanistic support for the decision to include an additional 2-fold protection factor for young populations in the recent US EPA VC risk assessment.
For More Information Contact:
James A Swenberg
University of North Carolina-Chapel Hill
Department of Environmental Sciences and Engineering
253C Rosenau Hall, CB# 7431
Chapel Hill, North Carolina 27599-7431
To learn more about this research, please refer to the following sources:
- Morinello EJ, Ham AL, Ranasinghe A, Nakamura J, Upton PB, Swenberg JA. 2002. Molecular dosimetry and repair of N2,3-ethenoguanine in rats exposed to vinyl chloride. Cancer Res 62:5189-5195. PMID:12234983
- Morinello EJ, Koc H, Ranasinghe A, Swenberg JA. 2002. Differential induction of N2,3-ethenoguanine in rat brain and liver after exposure to vinyl chloride. Cancer Res 62:5183-5188.
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