Dose-Response Relationships for N7-(2-Hydroxyethyl)Guanine Induced by Low-Dose [ 14 C]Ethylene Oxide: Evidence for a Novel Mechanism of Endogenous Adduct Formation Debbie A. Marsden, 1 Donald J.L. Jones, 1 Robert G. Britton, 1 Ted Ognibene, 2 Esther Ubick, 2 George E. Johnson, 3 Peter B. Farmer, 1 and Karen Brown 1 1 Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom; 2 Lawrence Livermore National Laboratory, Livermore, California; and 3 Swansea University, Swansea, United Kingdom Abstract Ethylene oxide (EO) is widely used in the chemical industry and is also formed in humans through the metabolic oxida- tion of ethylene, generated during physiologic processes. EO is classified as a human carcinogen and is a direct acting alkylating agent, primarily forming N 7-(2-hydroxyethyl)gua- nine (N 7-HEG). To conduct accurate human risk assessments, it is vital to ascertain the relative contribution of endoge- nously versus exogenously derived DNA damage and identify the sources of background lesions. We have therefore defined in vivo dose-response relationships over a concentration range relevant to human EO exposures using a dual-isotope approach. By combining liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography- accelerator mass spectrometry analysis, both the endogenous and exogenous N 7-HEG adducts were quantified in tissues of [ 14 C]EO-treated rats. Levels of [ 14 C]N 7-HEG induced in spleen, liver, and stomach DNA increased in a linear manner from 0.002 to 4 adducts/10 8 nucleotides. More importantly, the extent of damage arising through this route was insignificant compared with the background abundance of N 7-HEG naturally present. However, at the two highest doses, [ 14 C]EO exposure caused a significant increase in endogenous N 7-HEG formation in liver and spleen, suggesting that EO can induce physiologic pathways responsible for ethylene generation in vivo and thereby indirectly promote N 7-HEG production. We present evidence for a novel mechanism of adduct forma- tion to explain this phenomenon, involving oxidative stress and1-aminocyclopropane-1-carboxylicacidasapotentialbio- synthetic precursor to ethylene in mammalian cells. Based on the proposed pathway, N 7-HEG may have potential as a bio- markerofcellularoxidativestress. [CancerRes2009;69(7):3052–9] Introduction Ethylene oxide (EO) is a widely used industrial intermediate in the manufacture of chemicals and is used as an agricultural fumigant and sterilizing agent, particularly for medical equipment and heat-sensitive goods (1). It is estimated that in the U.S. health care sector, as many as 325,000 people are exposed directly or incidentally to EO in the workplace (2). Whereas initial epidemi- ology studies suggested links between an elevated risk of leukemia and stomach cancer and occupational exposure to EO (3–5), many subsequent reports are inconclusive or contradictory about the ability of EO to induce specific cancers or increase cancer-related mortality (6–9). Despite the conflicting epidemiologic evidence, EO is classified by the IARC as carcinogenic to humans, primarily based on results from animal carcinogenicity studies, the fact that it is a direct alkylating agent that elevates mutation frequencies in rodent models, and evidence of chromosomal damage in peripheral blood lymphocytes of exposed workers (10). The mutagenicity and carcinogenicity of EO is attributed to reaction with DNA, leading to the formation of multiple 2- hydroxyethyl adducts (11, 12). The most abundant product, N 7-(2- hydroxyethyl)guanine (N 7-HEG), readily depurinates, leaving abasic sites with miscoding potential (13, 14). Measurement of N 7-HEG in animal or human cellular DNA can provide a valuable biomarker of total EO exposure at the target site, vital information for risk assessment purposes. A confounding factor in evaluating the risks associated with EO inhalation is the fact that ethylene is also generated in vivo during normal physiologic processes and can be converted to the epoxide by cytochrome P 450 2E1 (15). Humans are therefore continually exposed to EO, as illustrated by detectable N 7-HEG at levels of f1 to 10/10 7 nucleotides in lymphocytes isolated from people not knowingly in contact with EO (16–18). Physiologic sources of ethylene are believed to include methionine oxidation, lipid peroxidation, and the metabolizing activity of intestinal bacteria (19–21); however, the mechanisms of formation in mammalian systems have not been defined in any of these cases and the origins of endogenous N 7-HEG adducts have never been directly shown. For genotoxic carcinogens such as EO, the current regulatory stance assumes that a linear relationship exists between exposure, the formation of DNA lesions, and subsequent conversion into mutations, although measurable increases in mutagenic events are only associated with relatively high doses (22, 23). Consequently, demonstration that a chemical is able to form DNA adducts at high exposures is often taken as sufficient evidence for carcinogenic potential at lower doses. However, nothing is actually known about the dose-response relationships for occupational or environmen- tally generated EO at the low concentrations humans are exposed to. Furthermore, this position fails to recognize that practical risk thresholds may be apparent for certain genotoxic agents where high levels of structurally identical endogenous adducts also exist (24). To assess the true risk associated with inhaled EO, distinct from that presented by ubiquitous background DNA damage caused by endogenous EO, it is necessary to distinguish between the two types of lesions and ascertain the relative contribution of damage arising from the different sources. Sensitivity limitations and an inability to differentiate identical adducts formed by multiple Requests for reprints: Karen Brown, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, United Kingdom. Phone: 44-116-223-1851; Fax: 44-116-223-1855; E-mail: kb20@le.ac.uk. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-4233 Cancer Res 2009; 69: (7). April 1, 2009 3052 www.aacrjournals.org Research Article