Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure.

S.Holt 0 T.-Y.Yen 0 R.Sangaiah 0 J.A.Swenberg 0 0 Department of Environmental Sciences and Engineering and Curriculum in Toxicology, University of North Carolina , Campus Box 7400, Rosenau Hall, Chapel Hill, NC 27599-7400, USA 1To whom correspondence should be addressed Email: The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (eA), 3,N4-ethenocytosine, 1,N2ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of e A in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic e A standard. This compound was confirmed by mass spectral analysis. e A was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of e A reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of e A, the exact source of the adduct is unknown. When rats were administered e A i.v., ~10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of e A may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools. - The chemical modification of DNA by carcinogens appears to be a critical step in the development of certain cancers (1). Quantitation of DNA adducts provides important information on the biologically effective dose of a carcinogenic agent, thus providing a better assessment of human exposure to environmental or endogenously formed carcinogens. The etheno DNA adducts are of particular concern since they are biologically relevant lesions resulting from exposure to industrial agents that are known human carcinogens, such as Abbreviations: 1,N2-e G, 1,N2-ethenoguanine; APNG, alkylpurine-DNA-Nglycosylase; BER, base excision repair; CAA, chloroacetaldehyde; CEO, chloroethylene oxide; e A, 1,N6-ethenoadenine; e dA, ethenodeoxyadenosine; e Ado, ethenoadenosine; HPLC, high performance liquid chromatography; IAC, immunoaffinity chromatography; LCMS/ESI/MS, liquid chromatography mass spectrometry/electrospray ionization/mass spectrometry; LPO, lipid peroxidation; MPG, N-methylpurine-DNA glycosylase; PBS, phosphate-buffered saline; SIM, selected-ion-monitoring; VC, vinyl chloride monomer. vinyl chloride (VC). These adducts are also formed endogenously, making them relevant to the population as a whole (2 4). Chronic exposure to VC is known to cause angiosarcoma of the liver in humans and animals (5,6). It is recognized that the carcinogenic and hepatotoxic effects of VC, and other vinyl halides, are not elicited directly by themselves, but through their P450 2E1-mediated biotransformation to extremely reactive intermediates (7). In the case of VC, a halooxirane, chloroethylene oxide (CEO), is formed and can further rearrange to the corresponding haloacetaldehyde, chloroacetaldehyde (CAA). CEO reacts with DNA more readily (8,9) and is more mutagenic than CAA (1013). Both are electrophilic species capable of producing the etheno adducts 1,N6-ethenoadenine (e A), 3,N4-ethenocytosine (e C), N2,3ethenoguanine (e G), and 1,N2-ethenoguanine (1,N2-e G). The cyclic ethenobases exhibit miscoding properties (1418) implicating them as critical promutagenic lesions responsible for vinyl halide carcinogenesis. DNA repair represents a major defense mechanism for the cell against the incorporation of mutations into the genome. One of the major pathways for repair of chemically induced DNA damage depends on the excision of altered bases by DNA glycosylases (base excision repair, BER). Subsequent to BER, excised DNA adducts are excreted into the urine. In vitro experiments have shown the etheno adducts to be released by a partially purified human etheno A-binding protein and cell-free extracts containing the cloned human N-methylpurineDNA glycosylase [alkylpurine-DNA-N-glycosylase, APNG (19)]. Recently, it was concluded that e C is released by a different glycosylase than e A in cell extracts of APNG-null mice (20). In addition, cell-free extracts from tissues of APNGnull mutant mice show no activity toward e A, whereas extracts from wild-type mice excised e A, indicating that e A is a substrate for APNG repair in mice (21). To our knowledge, there have been no reports on the excretion of etheno adducts into the urine. It has been postulated that DNA adducts excreted in urine can provide valuable information about human exposure to alkylating carcinogens (22). Methods based on urinary markers for use in human biomonitoring studies are currently being developed and include physicochemical methods, such as mass spectrometry, and immunochemical procedures. These techniques have proven useful in showing the urinary excretion of certain aflatoxin and various methylpurine adducts in urine following carcinogen exposure to rats and humans (2227). The development of an analytical method for monitoring an etheno adduct, e.g. e A, in urine would provide the basis for a non-invasive biomarker of etheno adduct formation. This paper describes the procedure for purifying e A by immunoaffinity chromatography (IAC) prior to high performance liquid chromatography (HPLC) and its application to the analysis of e A in rodent urine. In order to confirm whether the compound detected by IACHPLC was in fact e A, we used liquid chromatography/mass spectrometry (LC/MS) to provide struc Fig. 1. Experimental scheme for e A analysis. tural characterization as well. e A was selected as the most likely etheno adduct to be detected, based on the facts that it is formed in the greatest amount (9) and has the most rapid repair (19). Materials and methods Chloroethylene oxide synthesis Briefly, the preparation of CEO was based on the photochlorination of ethylene oxide (28). In a typical preparation, 5 ml ethylene oxide was condensed into a 15 ml graduated centrifuge tube and transferred by cannula to a 25 ml threenecked flask equipped with a dry-ice cold finger condenser. t-Butylhydrochlorite (1.5 ml) was added and the reaction irradiated with a 200 W incandescent bulb for 1 h in a 05C bath. The product was distilled under reduced pressure (20 mm) at a bath temperature of 50C and collected as a 1:1 mixture of CEO:t-butyl alcohol in a receiver cooled by a dry-ice bath. The exact composition of the collected p (...truncated)