Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Mutagenicity of DNA adducts derived from ethylene oxide exposure in the pSP189 shuttle vector replicated in human Ad293 cells
Introduction
Ethylene oxide (EO; CAS No. 75-21-8) is a widely used industrial intermediate in the manufacture of chemicals and a small fraction of that produced annually is employed as an agricultural fumigant and sterilizing agent, particularly for medical equipment and heat-sensitive goods. It is estimated that in the US health care sector alone as many as 325,000 people are exposed directly or incidentally to EO in the workplace [1]. Early epidemiology studies suggested links between occupational exposure to EO and an elevated risk of leukaemia and stomach cancer [2], [3], [4]. However, many subsequent reports have failed to demonstrate that EO induces specific human cancers or increases cancer-related mortality, and there is ongoing debate surrounding the risks associated with low exposures to this compound [5], [6], [7], [8].
Despite the conflicting epidemiological evidence, EO is classified by the IARC as carcinogenic to humans (Group I). This categorization is based to a large extent on results from animal carcinogenicity studies, the fact that it is a direct alkylating agent that elevates mutation frequencies in rodent models, and the evidence for chromosomal damage in peripheral blood lymphocytes of exposed workers [9]. The mutagenicity and carcinogenicity of EO is attributed to reaction with DNA, which leads to the formation of multiple 2-hydroxyethyl (HE) DNA adducts [10], [11], [12]. Reaction of EO with naked DNA in vitro affords N7-HEGuanine (N7-HEG) as the predominant product, accounting for ∼95% of the total adducts formed. Minor amounts of N3-(2-hydroxyethyl)-2′-deoxyadenosine (N3-HEA) and O6-HEdG have been identified from this interaction in vitro, whilst 2-hydroxyethylation also occurs at the N1 and N6 positions of adenine, and the N3 group of cytosine and thymine [11], [12].
Chronic 48-week inhalation exposure to relatively high concentrations of EO (100 and 200 ppm) produces elevated lacI mutation frequencies in the bone marrow of transgenic mice [13], with a specific increase in AT → TA transversions detected in bone marrow cells. It has been proposed that this distinct mutational response is due to the presence of adenine and thymine HE adducts. In contrast, shorter 4-week periods of exposure failed to increase the incidence of lacI point mutations in spleen, bone marrow or testicular seminiferous tubules of mice, but a significant increase was observed in the lung, a target organ in this species [14]. In the same study, EO induced a dose-related increase in the occurrence of Hprt mutant T lymphocytes isolated from spleen tissue [15], prompting the authors to suggest that following short-term exposure the primary mechanism of genotoxicity involves large deletions and chromosomal mutations, which are not detected by the lacI phage-based assay [13]. However, the underlying mechanisms that could account for such differences between short-term and chronic dosing are unknown. A high incidence of K-ras mutations has also been detected in EO-induced lung, Harderian gland (HG), and uterine neoplasms from B6C3F1 mice [16]. The prominent targeting of GC base pairs in the lung and HG tumours was taken as evidence that adduction of guanine and subsequent K-ras mutation is a likely mechanism of tumourigenesis in these mice. Importantly, the actual lesions responsible for EO-induced mutations in vivo have not yet been identified and very little is known regarding the role of the quantitatively minor adduction products.
There is presently no evidence that the most abundant product of EO alkylation, N7-HEG is directly promutagenic, although it does readily depurinate, leaving abasic sites, which are capable of inducing base substitutions, frameshifts or large deletions if replication occurs before they are repaired [17], [18]. However, the generation of AP sites from N7-HEG does not seem to be a primary mechanism for EO-induced mutagenesis; inhalation exposure does not result in the accumulation of apurinic (AP) sites in target organs of treated rats or cause a biologically significant enhancement in the expression of genes involved in base excision repair, suggesting other types of damage are more likely to be accountable [19]. This study is part of a program of work aimed at determining the biological relevance of low levels of DNA adducts derived from EO exposure. We have investigated the dose–response relationships for EO-induced DNA damage and mutational events occurring in human cells using the supF forward mutation assay with a view to determining whether there is a level of tolerable DNA damage, below which significant increases in mutations are not induced, and examining the role of various HE-DNA adducts.
Section snippets
Shuttle vector plasmid, bacterial strain, and cell lines
The plasmid pSP189 (4952 base pairs) containing the supF target gene (85 base pairs) and E. coli strain MB 7070 were gifts from M. Seidman, National Institute of Aging, NIH, Baltimore, MD, USA. Human embryonic adenovirus-transformed kidney cells (Ad293) were cultured from cells previously provided by Dr. A. Dipple, National Cancer Institute, Frederick, MD. Ad293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Sigma) at 37 °C in 5% CO2.
Study 1: dose–response relationships
Aliquots of pSP189 plasmid incubated with low concentrations of EO (10–2000 μM) in Study 1 were analysed by our validated LC–MS/MS method prior to use in the supF assay. Of the five different HE adducts measurable by this approach, only the N7-HEG adduct was detectable in plasmid samples. As would be predicted, the levels of this lesion increased in a dose-dependent, linear manner above the background damage present in control untreated plasmid, which averaged approximately 5 adducts/106
Discussion
The supF assay and related adaptations have previously been used to demonstrate the mutagenicity of damage formed by a wide variety of genotoxic agents in mammalian and bacterial cells, including bulky adducts, small alkyl lesions such as cyclic etheno adducts, O6-methyl-and O6-ethyl-2′-deoxyguanosine, and oxidative DNA damage [24], [25], [26], [27], [28], [29]. More recently, it was employed to support a lack of mutagenicity for acrolein in both repair proficient and deficient human
Conflict of interest
None.
Acknowledgements
We thank Dr. Michael Festing for help with the statistical analysis and Dr Didier Gasparutto (Laboratoire des Lésions des Acides Nucléiques, CEA, Grenoble, France) for kindly supplying the O6-HedG standard. This work was funded by the Lower Olefins Sector Group and the Ethylene Oxide and Derivatives Sector Group of the European Chemical Industry Council (Cefic) and the Olefins Panel of the American Chemistry Council.
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